Enhanced MSC preparation

ABSTRACT

The present invention provides preparations of MSCs with important therapeutic potential. The MSC cells are non-primary cells with an antigen profile comprising less than about 1.25% CD45+ cells (or less than about 0.75% CD45+), at least about 95% CD105+ cells, and at least 95% CD166+ cells. Optionally, MSCs of the present preparations are isogenic and can be expanded ex vivo and cryopreserved and thawed, yet maintain a stable and uniform phenotype. Methods are taught here of expanding these MSCs to produce a clinical scale therapeutic preparations and medical uses thereof.

CLAIM TO PRIORITY

This application is a divisional of U.S. patent application Ser. No.14/138,265, filed Dec. 23, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/267,363, filed Oct. 6, 2011, now abandoned andclaims the benefit of priority from U.S. Provisional Patent ApplicationNo. 61/391,452 entitled “Enhanced MSC Preparations,” filed on Oct. 8,2010, and U.S. Provisional Application No. 61/391,482, entitled“Nanoparticle-Loaded Cells,” filed on Oct. 8, 2010, the contents ofwhich are incorporated by reference in their entireties.

This application incorporates by reference, the International PCTApplication No. PCT/US2011/055072 entitled “Enhanced MSC Preparations,in the office of McAndrews, Held and Malloy, which is incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention generally relates to therapeutically effectivepreparations of mesenchymal stem cells (MSCs), methods for manufacturingsuch preparations, methods of screening and selecting such preparations,and therapeutic uses thereof.

BACKGROUND

One of the major problems in stem cell therapy is the lack ofunderstanding of the therapeutic properties of stem cells, and how thesetherapeutic properties are influenced by manufacturing methods.

Stem cells are populations of cells found in embryos, fetuses, and adulttissues that are capable of self-renewal in undifferentiated forms andregain the capability of toti-, pluri- or multi-potentialdifferentiation in conditioned environments.

Use of the term “stem” cell has generally been reserved for those cellspossessing the ability to self-replicate and give rise to daughter cellswhich undergo a unidirectional, terminal differentiation process. Threeadult tissues in which stem cells have been extensively studied includethe epidermis, gastrointestinal epithelium, and the hematopoieticcompartment of bone marrow. Of these, hematopoietic stem cells areperhaps the best characterized, and are noted for their ability to giverise to multiple cellular phenotypes through lineage progression ofdaughter progenitor cells.

The bone marrow stroma was originally thought to function mainly as astructural framework for the hematopoietic component of the marrow.Subsequently, it has become well established that the stroma consists ofa heterogeneous population of cells, a subset of which exerts bothpositive and negative regulatory effects on the proliferation anddifferentiation of hematopoietic stem cells (HSC) in the marrow througha combination of physical and chemical signals. The stroma also containsother non-hematopoietic cells termed mesenchymal stem cells (MSC), whichare capable of both self-renewal and differentiation into osteoblasts,adipocytes, myoblasts and chondroblasts. The number of HSCs in bonemarrow is about 10-100 times greater than that of MSCs. MSCs also giverise to a variety of mature cell types via a step-wise maturationprocess similar to hematopoiesis, termed mesengenesis. Functions thathave been attributed to MSCs include, for example, the daily control ofinflammation, immune response, hematopoiesis and organ integrity.

Despite the features ascribed to MSC populations by their in vitrodifferentiation capabilities, the mechanisms governing theirproliferation and multi-lineage differentiation capacity have beenpoorly understood. At the clonal level, there is little evidence for MSCself-renewal, therefore these cells might be termed multipotentprogenitor cells. One of the greatest obstacles in the study of MSCbiology is the heterogeneity of studied cell populations (Baksh et al.(2004) J Cell Mol Med 8, 301-16). For example, Pittenger et al. ((1999)Science 284, 143-7) found that the majority of human bone marrow derivedMSCs are not pluripotent, while Kusnestov et al., ((1997) J Bone MinerRes 12, 1335-47) showed that only 58.8% of human MSCs had in vivoosteogenic potential. Others have shown that within the adipose derivedpopulations of MSCs, cells with multi-lineage differentiation capabilityco-exist with single lineage committed cells (Zuk et al. (2002) Mol BiolCell 13, 4279-95). Others have reported pluripotent progenitor cells(Jiang et al. (2002) Nature 418, 41-9).

Ex vivo preparations of bone marrow aspirates can show a great diversityof cell types. Even when such preparations are enriched for MSCs (e.g.by adherence), there is a remarkable diversity and heterogeneity.Methods exist in the art to enrich and expand such MSC's in culture,nonetheless, heterogeneity is observed at biochemical, genetic, andphenotypic levels.

Even so-called “pure MSC” preparations demonstrate heterogeneity withvariation of therapeutic effect, potency, differentiation capacity,mitotic activity, and so forth. For example, MSCs are known to undergophenotypic rearrangements during ex vivo manipulations, losingexpression of some markers while acquiring new ones (Augello et al. “TheRegulation of Differentiation in Mesenchymal Stem Cells” HUMAN GENETHERAPY 21:1226-1238 (October 2010)). Depending on culture conditions,various MSC subsets are preferentially expanded in culture, differing,for example, in expression of surface markers and other proteins,differentiation capacity, proliferation, and morphology (Baksh et al.“Adult mesenchymal stem cells: characterization, differentiation, andapplication in cell and gene therapy,” J. Cell. Mol. Med. Vol 8, No 3,2004 pp. 301-316; Bobis et al. “Mesenchymal stem cells: characteristicsand clinical applications,” FOLIA HISTOCHEMICA ET CYTOBIOLOGICA. Vol.44, No. 4, 2006; pp. 215-230). Donor variability and donor tissue sitealso contribute to differences between preparation of cells (Zhukarevaet al. “Secretion profile of human bone marrow stromal cells: donorvariability and response to inflammatory stimuli,” Cytokine, 2010,Volume: 50, Issue: 3, Pages: 317-321; US 2009/0010896).

Thus, it is clear that various observations attributed to “MSCs” byvarious laboratories are very likely describing different subsets of MSCpopulations, even when populations share certain functional propertiestypical of MSCs in general such as proliferative or differentiationcapacity. For example, Xiao et al. describe a culture of bone marrowderived MSCs that has undergone 20 population doublings, exhibiting anantigen profile of less than 90% CD105+ cells and less than 85% CD166+cells (Xiao et al. “Clonal Characterization of Bone Marrow Derived StemCells and Their Application for Bone Regeneration” Int J Oral Sci, 2(3):127-135, 2010).

Changes of MSC cultures with passage (i.e. ex vivo expansion) are alsowell recognized. For example, in U.S. Pat. No. 5,486,359, Caplandescribes that in his MSC preparations, early passaged cells (1st-2ndpassages) gave more bone formation than late passaged cells (4th-6thpassages).

This heterogeneity can be explained, in part, by the hypothesis thatMSCs, with the ability to self-renew and differentiate into multiplelineages, are only a small sub-population of the pool of MSCs and theremainder of the mixed population consists of cells at various stages ofdifferentiation and commitment. Adding to the complexity ofheterogeneity within a single MSC population is the variety of tissuesfrom which MSC have been harvested and the variety of techniques thathave been utilized in their isolation and propagation (Gronthos et al.(2001) J Cell Physiol 189, 54-63; Peister et al. (2004) Blood 103,1662-8). Given these variabilities, populations and sub-populations ofso-called MSCs have not yet been systematically compared.

WO 2007/123363 (Choung et al.) describes an MSC preparation that appearsto be enriched for CD105+ cells and CD45− cells. However, Choung et al.does not teach an MSC preparation that is pure for CD166+ cells, CD105+cells, and CD45− cells and does not teach a clinical scale MSCpreparation containing a billion MSCs. Further, Choung et al. does notprovide teachings of other technical features such as TNFRI expression,immunosuppression by inhibition of IL-2Rα expression, resilience tocryopreservation, a capacity for adipogenic, chondrogenic, andosteogenic differentiation after passage expansion.

US 2009/0010896 (Centeno et al.) describes a preparation of primary MSCsthat are enriched for CD166+ cells, CD105+ cells, and CD45− cells.However, such a primary culture does not provide clinical scale MSCnumbers in the billions. Further, like Choung et al., Centeno et al.lacks teaching of other technical features useful for therapeutictreatment.

Choung et al. and Centeno et al. illustrate a problem facing the skilledartisan. The art is replete with reports of poorly characterizedpreparations based on a mixed variety of cellular phenotypes derivedfrom a mixed variety of manufacturing techniques. While there is atendency among some to combine teachings from such reports, suchcombinations can lead to false conclusions when the reports representdifferent MSC populations. To advance the understanding of MSC biologyand therapy, it will be important to fully characterize the phenotype ofMSCs in a preparation and to recognize heterogeneity where it exists.

What is needed in the art is the ability to manufacture uniformpreparations of MSCs in numbers sufficient for one or moretherapeutically-effective dose, having a reproducible therapeuticaction, and having a phenotype that is stable during ex vivo expansionand following cryogenic preservation. Also needed are such preparationsthat are isogenic, minimizing certain adverse effects associated withallogeneic transplantation.

SUMMARY OF THE INVENTION

Preparations of mesenchymal stem cells (MSCs) have now been discoveredwith enhanced therapeutic potential and with a structural and functionalphenotype that is stable in culture. Surprisingly, MSCs in thesepreparations can be expanded to provide a therapeutic dose; evenclinical scale preparations can now be made.

MSCs of the present preparations can be expanded ex vivo andcryopreserved and thawed, yet maintaining a stable and uniformphenotype.

The MSCs of the present invention are non-primary MSCs, having anantigen profile comprising less than about 1.25% CD45+ cells (or lessthan about 0.75% CD45+), at least about 95% CD105+ cells, and at leastabout 95% CD166+ cells.

MSCs of the present preparation have a phenotype characterized by one ormore of the following properties: TNFR1 expression, inhibition of IL2Rα(e.g. in a PBMC assay), greater than 70% viability after freeze-thaw,ability to maintain phenotype following ex vivo expansion, capacity fordifferentiation, and isogenic (i.e., derived from a single donor).

In one embodiment, the MSC preparation has a combination of unexpectedtechnical features, namely: a) it comprises at least about 1 billion(1×10⁹) cells; b) it has an antigen profile comprising less than about0.75% CD45+ cells, at least about 95% CD105+ cells, and at least about95% CD166+ cells; c) it comprises TNFRI in an amount of at least about13 pg (e.g. about 13 pg to about 44 pg) of TNFRI per million cells; d)the MSCs have a capacity for adipogenic, chondrogenic, and osteogenicdifferentiation; e) the MSCs are resilient to cryopreservation, i.e.after a freeze-thaw cycle, at least about 70% of the MCSs are viable, asassessed by dye exclusion; f) when mixed with PBMCs at a ratio of about5 PBMCs per MSC, the MSCs are capable of inhibiting IL2Rα expression byCD3/CD28-activated PBMCs cells by at least about 30%, relative to acontrol; g) it generally contains less than about 55 μg/mL BSA and lessthan about 42 μg/mL trypsin; h) it is substantially free of pathogens(e.g. endotoxin, bacteria, fungi, and viruses); and i) the MSCs have anormal karyotype. Surprisingly such an MSC preparation can comprise acell number of at least about any of: 10 billion, 100 billion, 500billion, or 1 trillion cells, even when expanded from a single bonemarrow donation.

Preparations of the above embodiment can be obtained by screeningpreparations made by various culture techniques known in the art or madeby screening preparations made by methods including one or more of thesteps of ex vivo expansion taught herein. Surprisingly, however, theinventors have discovered a novel method that involves a series of stepsthat result in consistent MSC preparations of the present invention, asdetailed herein, for example in Example 1 through Example 41.Accordingly, the invention also provides a collection of preparationswith low variability between preparations.

The invention also provides a method of preparing, screening, orselecting preparations.

The invention also provides a method of treatment comprisingadministering MSCs from a preparation to a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

As used here, the following definitions and abbreviations apply.

“BMA” means bone marrow aspirate.

“Clinical scale” as it refers to a present MSC preparation, means apreparation that contains more than one therapeutic dose. Optionally, aclinical scale preparation is derived from a single donor. For example,a clinical scale preparation of the present invention can comprise8,000±10% or ±20% doses of 125 million MSCs (or 100 million viable MSCsafter a freeze-thaw cycle). Optionally, a clinical scale preparation ofthe present invention can comprise 10,000±10% or ±20% doses of 125million MSCs (or 100 million viable MSCs after a freeze-thaw cycle).

“Freeze-thaw cycle” or “cryoprotective freeze-thaw cycle” meanscryogenic freezing followed by thawing and in vitro culturing underconditions to preserve viability, especially as taught here according tothe present invention.

“Therapeutic dose” is an amount of MSCs that, when administered to asubject in need thereof in a single administration (or optionally infractional amounts within a given period), can result in a clinicallyrelevant response. A clinically relevant response is any direct orindirect indicator of a positive therapeutic response (irrespective ofthe eventual outcome). Methods of determining therapeutic response arewell known in the art. By way of example, a therapeutic dose is about100 million MSCs or about 125 million MSCs. Other useful therapeuticdoses include about 2 million MSCs per kg, about 8 million MSCs per kg,and about 2-8 million MSCs per kg, for example, as detailed in Example42 through Example 45.

“Examplary” (or “e.g.,” or “by example”) means a non-limiting example.

“Isogenic” means that cells of a given population are autologous to eachother, i.e., derived from the same donor. The term isogenic is intendedto include, for example, natural variation in genetic makeup of cellsderived from the same donor.

“n±x %” means a range extending from [n−(x %·n)] to [n+(x %·n)]. Such aphrase is not intended to set forth error or precision in measurement.

“P_(n)”, where “n” is an integer, refers to the number of cells that aculture has been passage. P1 is the first passage after cells are platedfrom the bone marrow aspirate (primary culture).

The present invention provides a preparation comprising a uniformpopulation of MSC cells in sufficient numbers to have a clinicallysignificant effect and being greatly enriched for a subset of MSCs witha remarkable therapeutic potential. Surprisingly, it has been discoveredthat such a preparation can be further expanded ex vivo to producemultiple therapeutic doses, even from a single donor.

Overview of MSC Preparations.

An MSC preparation according to the present invention is an MSCpreparation that is 95% homogeneous with respect to being CD105 positiveand CD166 positive and being CD45 negative. This homogeneity persiststhrough ex vivo expansion; i.e. though multiple population doublings. Anexample of a useful embodiment is a preparation where the MSCs compriseless than about 1.25% CD45+ cells, at least about 95% CD105+ cells, andat least about 95% CD166+ cells, and contains at least one therapeuticdose. This homogeneity persists after cryogenic storage and thawing,where the cell also generally have a viability of about 70% or more.

MSC preparations of the instant invention express substantial levels ofTNFR1, for example greater than 13 pg of TNFRI per million MSCs. Thisphenotype is stable throughout ex vivo expansion and cryogenic storage.Indeed, the expression of levels of TNFRI in the range of about 13 toabout 179 pg (e.g. about 13 pg to about 44 pg) per million MSCs isassociated with a desirous therapeutic potential which also persiststhrough ex vivo expansion and cryopreservation.

Another feature of cells according to the present invention in highpotency, as defined by the ability of the cells to inhibit IL-2Rαexpression on T-cells. Typically, preparations according to the presentinvention can inhibit IL-2Rα expression by at least about 30%,alternatively at least about 35%, alternatively at least about 40%,alternatively at least about 45%, alternatively at least about 50%,alternatively at least about 55%, alternatively at least about 60.

Optionally, the preparations of MSCs of the present invention contain atleast one therapeutic dose (e.g., at least about 100 million cells orabout 125 million cells).

Unexpectedly, one embodiment of the present invention provides aclinical scale preparation providing multiple therapeutic doses (e.g.,expanded from a single donor).

Optionally, the preparations of the present invention, including, forexample, a clinical scale preparation, can be produced that have one ormore additional unexpected profiles that especially suit the preparationfor clinical use. For example, the preparations of the present inventionmay have a profile based on potency (e.g., TNFRI activity), resilienceto cryopreservation (e.g., viability after a freeze thaw cycle),differentiation capacity (i.e., the ability to differentiate into celltypes), retention of differentiation capacity, unexpected therapeuticutility as taught herein, or any combination of the aforementionedproperties.

Optionally, the preparations of the present invention comprise at least100 million cells, have an antigen profile of less than about 1.25%CD45+ cells, at least about 95% CD105+ cells, and at least about 95%CD166+ cells and the cells can be expanded ex vivo from passage 2 untilpassage 5 while maintaining population uniformity based upon the antigenprofile (i.e. less than about 1.25% CD45+ cells, at least about 95%CD105+ cells, and at least about 95% CD166+ cells).

An MSC preparation can be in any form. In one embodiment, a preparationis provided in a single volume or aliquot (e.g., in a single containercomprising the entire preparation). In another embodiment, a preparationis provided as a non-continuous preparation (e.g., the preparation isprovided as a plurality of spatially separate compositions). Optionally,a non-continuous preparation is provided in a plurality of vessels,wherein each of the plurality of vessels contains a portion of thepreparation. Optionally, the vessels are bags, or other useful storagecontainers. Optionally, the vessels are other therapeutic dosage form.Optionally, the vessels are culture vessels. Optionally, a continuous ornon-continuous preparation is present in a facility, such as an MSCmanufacturing facility.

Optionally, the MSCs in the preparation are isogenic (e.g., derived fromthe same donor).

Optionally, the majority of MSCs in the preparation have about the sameex vivo age.

Optionally, the majority of MSCs in the preparation are of about thesame generation number (i.e., they are within about 1 or about 2 orabout 3 or about 4 cell doublings of each other). Optionally, theaverage number of cell doublings in the present preparations is about 20to about 25 doublings. Optionally, the average number of cell doublingsin the present preparations is about 9 to about 13 (e.g., about 11 orabout 11.2) doublings arising from the primary culture, plus about 1,about 2, about 3, or about 4 doublings per passage (for example, about2.5 doublings per passage) as seen in the MSC preparations set forth inExample 30 and Example 32. Examplary average cell doublings in presentpreparations are any of about 13.5, about 16, about 18.5, about 21,about 23.5, about 26, about 28.5, about 31, about 33.5, and about 36when produced by about 1, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, and about 10 passages, respectively.

Optionally, the majority of MSCs in the preparation have about the samepassage number (e.g., they are within about 1 or about 2 or about 3 orabout 4 passages numbers of each other). Examplary passage numbers inpresent preparations are any of about 2 to about 5, about 2 to about 6,about 2 to about 7, about 2 to about 8, about 3 to about 5, about 3 toabout 6, about 3 to about 7, about 3 to about 8, about 4 to about 7,about 4 to about 8, about 4 to about 9, or less than any of about 10,about 11, or about 12.

Preparation Profiles

Surprisingly, it has been discovered that MSCs can be cultured andexpanded ex vivo to produce a preparation containing at least onetherapeutic dose (or optionally, multiple therapeutic doses constitutinga clinical scale preparation) with an antigen profile of less than about1.25% CD45+ cells, at least about 95% CD105+ cells, and at least about95% CD166+ cells (e.g., as set forth in Example 30 or Example 32).Additionally, the MSC preparations having such an antigen profile can beproduced that also have one or more other unexpected profiles thatespecially suit the preparation for clinical use For example, thepreparation may exhibit unexpected potency (e.g., TNFRI concentration oractivity as set forth in Example 30 or Example 32), resilience tocryopreservation (e.g., viability after a freeze thaw cycle as set forthin Example 30 or Example 32), differentiation capacity or retention anddifferentiation (e.g., the ability to differentiate into cell types asset forth in Example 30 or Example 32).

In some embodiments of the present technology, the preparations of MSCshave an antigen profile of at least about 95% CD166+ cells,alternatively at least about 95.5% CD166+ cells, alternatively at leastabout 96%, alternatively at least about 96.5%, alternatively at leastabout 97%, alternatively at least about 97.5%, alternatively at leastabout 98%, alternatively at least about 98.5%, alternatively at leastabout 99%, alternatively at least about 99.5% of CD166+ cells, or anyamounts in between, for example, in increments of about 0.01%, about0.025%, about 0.05%, about 0.075% about 0.1%, etc. In some embodiments,the MSCs of the present technology further include an antigen profile ofat least about 95% CD105+ cells, alternatively at least about 95.5%,alternatively at least about 96%, alternatively at least about 96.5%,alternatively at least about 97%, alternatively at least about 97.5%,alternatively at least about 98%, alternatively at least about 98.5%,alternatively at least about 99%, alternatively at least about 99.5% ofCD105+ cells, or any amounts in between, for example, in increments ofabout 0.01%, about 0.025%, about 0.05%, about 0.075%, about 0.1%, etc.In some embodiments, the MSCs of the present technology have an antigenprofile of less than about 1.75% CD45+ cells, alternatively less thanabout 1.7%, alternatively less than about 1.65%, alternatively less thanabout 1.6%, alternatively less than about 1.55%, alternatively less thanabout 1.5%, alternatively less than about 1.45%, alternatively less thanabout 1.4%, alternatively less than about 1.35%, alternatively less thanabout 1.3%, alternatively less than about 1.25%, alternatively less thanabout 1.2%, alternatively less than about 1.15%, alternatively less thanabout 1.1%, alternatively less than about 1.05%, alternatively less thanabout 1%, alternatively less than about 0.95%, alternatively less thanabout 0.9%. alternatively less than about 0.85%, alternatively less thanabout 0.8%, alternatively less than about 0.75%, alternatively less thanabout 0.7%, alternatively less than about 0.65%, alternatively less thanabout 0.6%, alternatively less than about 0.55%, alternatively less thanabout 0.5%, alternatively less than about 0.45%, alternatively less thanabout 0.4%, alternatively less than about 0.35%, alternatively less thanabout 0.3%, alternatively less than about 0.25%, alternatively less thanabout 0.2%, alternatively less than about 0.15%, alternatively less thanabout 0.1%, alternatively less than about 0.05%, alternatively less thanabout 0.04%, alternatively less than about 0.03%, alternatively lessthan about 0.01%, or any amounts in between, for example, in incrementsof about 0.001%, about 0.005%, about 0.01%, about 0.025%, about 0.05%,about 0.075%, about 0.1%, etc. In some embodiments, the antigen profileof the MSCs is about 95% or greater of CD166+ cells, about 95% orgreater of CD105+ cells, and 1.75% or less of CD48+ cells, preferablyabout 97.5% or greater of CD166+ cells, about 98% or greater of CD105+cells, and 1.48% or less of CD48+ cells. In one embodiment, theinvention provides a preparation, therapeutic dose, or a clinical scalepreparation having a quantity of MSCs and an antigen profile set forthin Example 30 or Example 32. The antigen profile is a useful indicatorof phenotypic purity. Methods for determining an antigen profile areprovided herein.

In one embodiment, the invention provides a therapeutic dose or aclinical scale preparation having a TNFRI profile of greater than about13 pg per million MSCs, or optionally about 13 pg to about 179 pg permillion MSCs (e.g., as set forth in Example 30 or Example 32) such asabout 13 pg to about 44 pg/million MSCs. Among other qualities, a TNFRIprofile is a useful indicator of potency of the preparation (e.g.,capacity for immunosuppression). The potency and activity in the TNFRIprofiles of the present invention are based on the relative amount ofTNFRI (pg per million MSCs) in the preparation (e.g., as set forth inExample 30 or Example 32). The activity of the preparation can beexpressed as the percent IL-2Rα expression inhibition onmitogen-stimulated white blood cells (e.g., as set forth in Example 32).Methods for determining relative TNFRI amount or aggregate cellularpreparation activity are provided herein.

In one embodiment, the invention provides a therapeutic dose or aclinical scale preparation having a favorable cryopreservation profile,i.e., favorable post-thaw viability. A cryopreservation profile of thepresent invention indicates the resilience of the cells tocryopreservation as the percentage of viable cells in the compositionafter a freeze thaw cycle. A cryopreservation profile indicates thepercentage of cells that are viable after a freeze-thaw cycle comparedto an unfrozen composition. Examplary post-thaw viabilities of presentpreparations are at least about 70%, about 75%, about 80%, about 82%, orabout 85% viable cells (e.g., as set forth in Example 30 or Example 32).Methods for determining viability after a freeze thaw cycle are providedherein.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation having a favorable differentiation profile (e.g., asset forth in Example 30 or Example 32). A differentiation profile of thepresent invention indicates the MSCs' capacity for each of osteogenic,chondrogenic, and adipogenic differentiation at a given passage numberor doubling number. Methods for determining differentiation capacity aredescribed herein.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation having a normal karyotype, i.e. a preparation havingcells with no chromosomal abnormalities, for example, as described inExample 37. Chromosomal abnormalities that can be screened optionallyinclude numerical and structural abnormalities, such as translocations,breaks, rings, markers and double-minutes.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation comprising MSCs with a spindle-shaped morphology orappearance (as opposed to star, flat, or round shape). Optionally, atleast about any of: about 80%, about 90%, or about 95% or greater of theMSCs exhibit a spindle-shaped morphology.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation free of pathogen contaminants (e.g. fungi, viruses,mycobacteria, and other pathogenic bacteria), for example, as detailedin Example 38.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation free of abnormalities in telomerase activity. Methodsof determining such are well known in the art.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation wherein the cells are non-senescent. Methods ofdetermining senescence are well known in the art, for example asdescribed by Wagner et al. (Wagner et al., “Replicative Senescence ofMesenchymal Stem Cells: A Continuous and Organized Process”, PLoS ONE.2008; 3(5): e2213; Wagner et al. “How to track cellular aging ofmesenchymal stromal cells?,” Aging (Albany N.Y.). 2010 April; 2(4):224-230). Optionally, non-senescent MSCs do not have elevated levels ofsenescent markers such as micro-RNA markers or protein markers (e.g.expression is similar to P1 MSC cultures, or within about 10%, or about25%, or about 50%). For example, hsa-mir-371, hsa-mir-369-5P,hsa-mir-29c, hsa-mir-499 and hsa-mir-217; GPNMB; RAMP; PERP; LY96;STAT1; and PRNP have all been shown to be up-regulated in senescent MSCs(Wagner et al.). Optionally, non-senescent cells exhibit typicalmorphology of non-senescent cells. For example, it has been shown thatsenescent MSCs enlarge and generate more vacuoles and cellular debriscompared to non-senescent cells (Wagner et al.). Optionally, the cellsremain non-senescent for at least 1, 2, or 3 additional populationdoublings.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation having an antigen profile, TNFRI profile,cryopreservation profile, and differentiation profile, as describedabove.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation having an antigen profile, TNFRI profile, karyotype,and differentiation profile, as described above. Optionally, thetherapeutic dose or clinical scale preparation further comprises acryopreservation profile, as described above.

In one embodiment, the invention provides a pathogen-free therapeuticdose or clinical scale preparation having an antigen profile, TNFRIprofile, and differentiation profile, as described above. Optionally,the therapeutic dose or clinical scale preparation further comprises acryopreservation profile, as described above. Optionally, thetherapeutic dose or clinical scale preparation further comprises anormal karyotype, as described above.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation having an antigen profile, TNFRI profile, telomeraseactivity, and differentiation profile, as described above. Optionally,the therapeutic dose or clinical scale preparation further comprises acryopreservation profile, as described above.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation having an antigen profile, TNFRI profile, morphology,and differentiation profile, as described above. Optionally, thetherapeutic dose or clinical scale preparation further comprises acryopreservation profile, as described above.

In one embodiment, the invention provides a therapeutic dose or clinicalscale preparation having an antigen profile, TNFRI profile,non-senescent state, and differentiation profile, as described above.Optionally, the therapeutic dose or clinical scale preparation furthercomprises a cryopreservation profile, as described above.

Clinical Scale Preparations

Surprisingly, a population of MSCs, e.g., derived from only a singledonor, can expanded to a clinical scale preparation having an antigenprofile taught herein (e.g., as detailed forth in Example 30 or Example32).

A clinical scale preparation of the present invention provides severalsuperior advantages. The availability of multiple, nearly identicaltherapeutic doses (i.e., within the variance taught here) allows morepredictable clinical responses. Subjects that respond to a therapeuticdose can be subsequently treated with reduced risk of toxicity. Asinformation is obtained for various conditions, dosing (frequency andamounts) can be adjusted to achieve better therapeutic outcomes.

Without being bound by theory, it is believed that the therapeuticallyeffective amount of cells is highly dependent on the nature of thepreparation itself, for example, the phenotypic purity, potency,resilience to cryopreservation, and/or differentiation capacity of cellsin the preparation. Unless indicated otherwise, a therapeuticallyeffective dose of MSC preparations of the present invention is about 100million cells or about 125 million cells. While the skilled artisan canreadily determine the number of the present MSCs required for atherapeutic dose, in general (depending upon the therapeutic need), sucha number can be any of about 50 million, about 100 million, about 250million, or about 500 million, or about 1×10⁶ cells for example. Otheruseful therapeutic doses include about 2 million MSCs per kg, about 8million MSCs per kg, and about 2 to about 8 million MSCs per kg, forexample, as detailed in Example 42 through Example 45.

Surprisingly, an MSC population derived from only a single donor can beexpanded to produce a clinical scale MSC preparation containing about atleast any of: about 2; about 8; about 11; about 36; about 50; about 150;about 220; about 690; about 960; about 1,000; about 2,000; about 3,000;about 4,200; about 5,000; about 6,000; about 7,000; or about 8,000therapeutic doses, wherein the preparation has the antigen profiledescribed above (e.g., as set forth in Example 30 and Example 32).

By way of example, one embodiment of the present invention provides anMSC preparation of about 1×10¹² or about 1×10¹²±about 10%, about 20%,about 30%, or about 40% MSCs, with the antigen profile described above.Such an MSC preparation is capable of providing, for example, about8,000±about 10%, about 20%, about 30%, or about 40% doses to a patientbefore or after a freeze-thaw cycle (e.g., a patient with an immunemodulatory disease such as GVHD) or, optionally about 10,000±about 10%,about 20%, about 30%, or about 40% doses to a patient before or after afreeze-thaw cycle. Such a clinical scale preparation is quiteremarkable, for example, because a single patient can be frequently andrepeatedly treated with doses of MSCs expanded from a single donation(e.g., bone marrow donation of 100 ml to 120 ml). For example, with theteachings provided herein, a single donation of bone marrow (e.g.,100-120 ml) can be used to produce a preparation of MSCs which can beused to treat a patient for many months or many years (i.e., lifelongtreatment).

With the teachings provided herein, the skilled artisan can now makeclinical scale MSC preparations having a desired number of MSCs with theantigen profiles taught herein. In general, the final number of MSCs ina preparation is dependent the starting number of primary MSCs (orvolume of bone marrow aspirate) and the expansion level. However, largeaspirate and culture volumes introduce a number of technical challenges.Successful culture of cells to provide clinical scale preparations ofthe instant invention can require a heavy burden on cell handling andsterile technique. Even the most well-established culture methods canimpart culture heterogeneity in a multitude of ways. A present clinicalscale preparation can optionally be obtained by serial passage expansionwhere each passage includes a step of splitting the previous cultureinto a plurality of cultures at a given ratio. Each passaging stepincreases the number of concurrent cultures in the preparation. By wayof example, the method detailed in Example 1 through Example 25 producesover 7,000 cell factories for a single isogenic preparation. It is quiteremarkable that, by following the teachings provided herein, one cansuccessfully produce clinical scale preparations having the instantpreparation profiles, e.g. antigen profile, TNFRI profile,cryopreservation profile, differentiation profile, and/or sterility(with respect to pathogens).

In one embodiment, a preparation is provided comprising at least about1×10⁹ MSCs (e.g. ≥P2 cells). In one embodiment, a preparation isprovided comprising at least about 4.5×10⁹ MSCs (e.g. ≥P2 cells). In oneembodiment, a preparation is provided comprising at least about 31×10⁹MSCs (e.g. ≥P3 cells). In one embodiment, a preparation is providedcomprising at least about 177×10⁹ MSCs (e.g. ≥P4 cells). In oneembodiment, a preparation is provided comprising at least about 8×10¹¹MSCs (e.g. ≥P5 cells). In one embodiment, a preparation is providedcomprising at least about 1×10¹² MSCs (e.g. ≥P5 cells). In oneembodiment, a preparation is provided comprising about 1×10⁹ to about1×10¹² MSCs (e.g. P1, P2, P3, P4, or P5 cells). In one embodiment, apreparation is provided comprising about 4.5×10⁹ to about 1×10¹² MSCs(e.g. P2, P3, P4, or P5 cells). In one embodiment, a preparation isprovided comprising about 31×10⁹ to about 1×10¹² MSCs (e.g. P3, P4, orP5 cells). In one embodiment, a preparation is provided comprising about177×10⁹ to about 1×10¹² MSCs (e.g. P4, or P5 cells). In addition tohaving the antigen profile taught herein, the preparations listed abovepreparations optionally have a TNFRI profile, cryopreservation profile,and/or differentiation profile taught herein. Optionally, thepreparations above retain the antigen profile after one or moreadditional population doublings. The invention also provides apreparation comprising any of: about 1×10⁹ to about 2×10¹² MSCs, about4.5×10⁹ to about 2×10¹² MSCs, about 31×10⁹ to about 2×10¹² MSCs, andabout 177×10⁹ to about 2×10¹² MSCs, about 1×10⁹ to about 3×10¹² MSCs,about 4.5×10⁹ to about 3×10¹², about 1×10⁹ to about 4×10¹² MSCs, about4.5×10⁹ to about 3×10¹², about 31×10⁹ to about 3×10¹² MSCs, about177×10⁹ to about 3×10¹² MSCs, about 31×10⁹ to about 4×10¹² MSCs, andabout 177×10⁹ to about 4×10¹² MSCs.

The number of cells in a preparation of the instant invention can beestimated by considering various factors such as the initial volume ofbone marrow aspirate (BMA) and number of population doublings (PD).

Table 1 shows examplary cell numbers in a clinical scale preparationbased on the number of PD and the initial volume of BMA.

TABLE 1 Example Cell Numbers MSCs-120 ml MSCs-70 ml MSCs-20 ml PD BMABMA BMA 30 3.20E+13 1.87E+13 3.11E+12 29 1.60E+13 9.33E+12 1.56E+12 288.00E+12 4.67E+12 7.78E+11 27 4.00E+12 2.33E+12 3.89E+11 26 2.00E+121.17E+12 1.94E+11 25 1.00E+12 5.83E+11 9.72E+10 24 5.00E+11 2.92E+114.86E+10 23 2.50E+11 1.46E+11 2.43E+10 22 1.25E+11 7.29E+10 1.22E+10 216.25E+10 3.65E+10 6.08E+09 20 3.13E+10 1.82E+10 3.04E+09 19 1.56E+109.11E+09 1.52E+09 18 7.81E+09 4.56E+09 7.60E+08 17 3.91E+09 2.28E+093.80E+08 16 1.95E+09 1.14E+09 1.90E+08 15 9.77E+08 5.70E+08 9.49E+07 144.88E+08 2.85E+08 4.75E+07 13 2.44E+08 1.42E+08 2.37E+07 12 1.22E+087.12E+07 1.19E+07 11 6.10E+07 3.56E+07 5.93E+06 10 3.05E+07 1.78E+072.97E+06Collections of Preparations

In one embodiment, the invention provides a collection (sometimes simplyreferred to as ‘a composition’) of at least three preparations, whereineach preparation comprises a therapeutic dose (or multiple doses as in aclinical scale preparation) of MSCs with an antigen profile taughtherein (e.g., as set forth in Example 30 or Example 32), wherein eachpreparation is derived from a different donor, and wherein the varianceof %0045+ cells is less than about 0.5%, optionally less than about0.1%, optionally less than about 0.05%, alternatively optionally lessthan about 0.4%, optionally less than about 0.3%, or optionally aboutless than 0.2%, the variance of % CD105+ cells is less than about 1%,optionally less than about 0.5%; and/or the variance of % CD166+ cellsis less than about 2.5%, optionally less than about 2%, optionally lessthan about 1% (e.g., as set forth in Example 34). Optionally, eachpreparation additionally comprises one or more other profiles of setforth in Example 30 or Example 32.

In one embodiment, the invention provides an MSC manufacturing facilitycomprising a collection of at least three preparations, wherein eachpreparation of the collection comprises an antigen profile taught herein(e.g., as set forth in Example 30 or Example 32), wherein eachpreparation is derived from a different donor, wherein the variance of %CD45+ cells is less than about 0.5%, optionally less than about 0.1%,the variance of % CD105+ cells is less than about 1%, optionally lessthan about 0.5%; and the variance of % CD166+ cells is less than about2.5%, optionally less than about 2%, optionally less than about 1%(e.g., as set forth in Example 34), and wherein greater than: about 50%,about 60%, about 70%, about 80%, about 90%, or about 95% of all clinicalscale MSC preparations (e.g., preparations having at least any of: about1×10⁹, about 5.5×10⁹, about 31×10⁹, about 177×10⁹, or about 8×10¹¹ MSCs)also have the antigen profile, for example, as described in Example 30or Example 32.

In one embodiment, the invention provides a method of makingtherapeutically acceptable MSCs comprising obtaining a bone marrowaspirate containing MSCs, expanding the number of MSCs to produce apreparation having at least one therapeutic dose (e.g. a clinical scalepreparation), and selecting the batch for therapeutic use if thepreparation has an antigen profile taught herein (e.g., as set forth inExample 30 or Example 32). Optionally, upon repeating said obtaining andexpanding steps a plurality of times to produce a plurality ofpreparations derived from different donors, greater than: about 50%,about 60%, about 70%, about 80%, about 90%, or about 95% of thepreparations have an antigen profile taught here, for example, asdescribed in Example 30 or Example 32.

Formulation

The MSC preparations of the present invention can optionally beformulated for pharmaceutical administration. For example, anypharmaceutically acceptable diluents can be combined with a therapeuticdose (e.g., to form a therapeutic product). Optionally, the therapeuticproduct comprises albumin, such as human serum albumin (HSA) or bovineserum albumin (BSA). Optionally, the therapeutic product comprises anelectrolyte solution, for example, to provide physiological osmolalityand pH (e.g., Plasma-LyteA, Baxter, Deerfield Ill.). Optionally, thetherapeutic product comprises a cryopreservative, such as DMSO.Optionally, during formulation of albumin (e.g., with acryopreservative, such as DMSO), the formulation is chilled beforemixing with albumin, for example, to prevent possible degradation ofalbumin by exothermic reaction.

In one embodiment, a therapeutic product comprises albumin, anelectrolyte solution, and a cryopreservative. Optionally, thetherapeutic product comprises about 0.1% to about 15% albumin (e.g.,about 1% to about 15% such as about 5%) by weight and about 5% to about20% cryopreservative by volume (e.g., about 10%). Optionally, thetherapeutic product comprising albumin is HSA (human serum albumin), theelectrolyte solution comprises Plasma-LyteA, and the cryopreservativecomprises DMSO.

In one embodiment, a therapeutic product comprises a therapeutic dose.

In one embodiment, a therapeutic product comprises a plurality oftherapeutic doses (e.g., about 8,000±about 10% or about 20% doses, or,optionally, about 10,000±about 10% or about 20% doses, and includeranges there between). In one embodiment, the therapeutic productcomprises a plurality of therapeutic doses derived from the same donor.

Manufacture

Surprisingly, a bone marrow aspirate containing only a small fraction ofthe MSCs of the present invention can be processed to produce apreparation containing a relatively uniform and reliable population ofMSCs, expanded to a sufficient quantity for a therapeutic dosage or fora clinical scale preparation. Such preparations have an antigen profiletaught herein (e.g., a preparation set forth in Example 30 or Example32), optionally having one or more profiles selected from the groupconsisting of potency, resilience to cryopreservation, differentiationcapacity and retention of differentiation capacity, as taught herein(e.g., as set forth in Example 30 or Example 32).

Generally, a donor is selected as taught here, a biological sample isobtained, the sample is screened (e.g., by methods taught here),acceptable samples are cultured to obtain adherent cells, the adherentcells are expanded under conditions that greatly enrich for the MSCswith desired phenotype (e.g., having an antigen profile taught herein).The culture is expanded to contain a number of cells for a therapeuticdose or for a clinical scale preparation. The preparation can becryopreserved at one or more passages during expansion.

An MSC preparation of the present invention can be prepared by obtaininga population comprising MSCs, expanding the number by passaging thepopulation one or more times, and selecting cells with aCD105+/CD166+/CD45− phenotype to provide a therapeutic dose or aclinical scale MSC preparation having an antigen profile taught herein(e.g., a preparation set forth in Example 30 or Example 32). In oneembodiment, the selection process comprises a step of culturing apopulation in the presence of a substrate and selecting cells thatadhere to the substrate. Optionally, the selecting step does notcomprise immunoselecting (e.g. antibody-based purification). Optionally,the selecting step consists essentially of culturing a population in thepresence of a substrate and selecting cells that adhere to thesubstrate, and optionally serially repeating said culturing andselecting steps one or more times.

As set forth below, the MSC preparations of the present invention can beproduced by a series of steps, including (1) obtaining living cells froma biological sample of bone marrow (e.g. a bone marrow aspirate); (2)combining the cells with growth medium on a surface suitable foradherent cells; (3) cultivating the cells in vitro to increase the cellnumbers; (4) expanding the cells numbers further through “passaging” (orsubculturing); (5) optional selecting preparations that have been“qualified” by structural and function features of the therapeutic MSCpreparations of the present invention; (6) optionally, cryopreservingthe cells; and (6) optionally thawing the cells for subsequenttherapeutic administration.

Bone marrow aspirates can typically be more than about 50 ml such asabout 100 ml to about 120 ml bone marrow. These cells are “seeded” inappropriate tissue culture medium containing factors that stimulate MSCgrowth relative to differentiation. Initial seeding concentrations canbe about 40 to about one million cells per cm² of culture dish, or about146 or about 146 plus and minus 50 or plus and minus 100 or in the rangeof about 50 to about 500 cells/cm² of culture dish. Primary cells areselected for adherence to an appropriate substrate surface andcultivated for one or more weeks (e.g., for about 21±3 days), removingthe non-adherent matter from the substrate surface by replacing themedium with a fresh medium and allowing the adherent MSCs toculture-expand.

Optionally, these primary cells can be further passaged to non-primarycells (e.g. removed from the culture surface and expanded intoadditional area) by seeding at a density of about 1,000 to about onemillion nucleated cells/cm² of culture dish (e.g. about 5,900 cells/cm²plus and minus about 1,200), and then culturing for additional days,e.g. about 14±about 2 days. In suitable embodiments, the primary cellsare grown to confluence, and in some instances are passaged to a secondculture of non-primary cells by seeding the primary cells from aconfluent primary cell culture in the second culture surface in anamount below confluence and growing the non-primary culture toconfluence. This method can be repeated for additional passages.

This ex vivo culture expansion can optionally comprise additionalpassaging (e.g., about 3, about 4, or about 5 cycles). The method cancomprise, for example, passaging a number of times to provide a fivepassage (P5) culture.

Donor Selection

In one embodiment, a population of MSCs is derived from a tissuespecimen containing MSCs (e.g., bone marrow). The donor can be any donorhaving MCS-containing tissue. Optionally, the donor is a human.Optionally, the donor is a living human. Optionally, the donor is not afetus. Optionally, the donor is in a post-natal stage of life.

In one embodiment, a donor is selected for tissue donation (e.g., bonemarrow donation) based on predetermined selection criteria. Optionally,a donor is determined to be eligible if the donor is free from riskfactors, shows no signs of infection due to relevant communicabledisease agents and infectious diseases, or is deemed physically fit toundergo the bone marrow aspirate procedure as per HCT/P regulations, forexample, as set forth in Example 2.

In one embodiment, a donor is a “qualified donor”. A qualified donor isa donor that is selected based on the results of one or more or allassessments/tests listed in Table 2. Optionally, a qualified donor is adonor having one or more of: a minimum of one successful donation, aprevious bone marrow cell count greater than about 2.0×10⁶ cells/mL, ahistory of negative infectious disease tests, a Body Mass Index (BMI)<about 30, and an age of about 18 to about 30 years.

In one embodiment, a qualified donor is negative or non-reactive forsome or all of the diseases set forth in Table 3. Optionally, the donoris screened for said diseases using the respective example assay/testset forth in Table 3.

Donor Tissue Source

In order to obtain the present preparations, it is necessary to obtainand selectively expand the MSCs from biological samples.

Bone marrow cells can be obtained from iliac crest, femora, tibiae,spine, rib or other medullary spaces. Optionally, bone marrow isobtained as soft tissue, for example, occupying the medullary cavitiesof long bones, some haversian canals, and spaces between trabeculae ofcancellous or spongy bone. Optionally, bone marrow is obtained as eitherof two types: red, which is found in all bones in early life and inrestricted locations in adulthood (i.e., in the spongy bone) and isconcerned with the production of blood cells (i.e., hematopoiesis) andhemoglobin; and yellow, which consists largely of fat cells andconnective tissue.

The collection of MSCs-containing tissue can be performed by any methodknown in the art.

In one embodiment, bone marrow is collected by aspiration, for example,as set forth in Example 3. Optionally, the method of collection involvesthe use of an anticoagulant, for example, heparin, as set forth inExample 3. Optionally, the bone marrow is collected from the iliaccrest.

In one embodiment, up to about 120 ml of bone marrow is collected from adonor, for example, as set forth in Example 3. In another embodiment, atleast about 60 ml or more of bone marrow is collected. Optionally, about60 ml (±about 10%, about 20%, or about 30%) of bone marrow is collectedfrom each side of the iliac crest. Optionally, about 80 ml to about 120ml or about 80 ml to about 160 ml is collected. Optionally, about 20 mlto about 40 ml, about 40 ml to about 60 ml, about 60 ml to about 80 ml,about 80 ml to about 100 ml, about 100 ml to about 120 ml, or about 120ml to about 120 ml of bone marrow is collected. Optionally, the bonemarrow is collected from the iliac crest. Optionally, the bone marrow iscollected from the iliac crest and a volume equal to about half thevolume in the ranges taught above is collected from each side thereof.

In one embodiment, donor tissue (e.g., BMA) is placed in a containerhaving a tubing harness or other means allowing for transfer to aprocessing device For example, the processing device may be in afunctionally closed system of processing steps comprising filling,sampling, and transferring of solutions, as set forth in Example 4.

Isolation of an MSC-Containing Population

An MSC-containing population can be isolated from MSC-containing tissue(e.g., bone marrow) by any method known in the art.

In one embodiment, isolation of an MSC-containing population comprises astep of isolating nucleated bone marrow cells (e.g., from BMA). Methodsfor the isolation of nucleated bone marrow cells are known in the art.Optionally, nucleated bone marrow cells are isolated by diluting bonemarrow (e.g., BMA) and separating nucleated bone marrow cells from othercells (e.g., red blood cells). Optionally, separating comprises allowingthe other cells to settle and extracting a layer of nucleated bonemarrow cells, for example, as set forth in Example 5.

In one embodiment, nucleated bone marrow cells (e.g., obtained asdescribed above) are washed and/or concentrated. Any composition knownfor washing can be used (e.g., a physiological salt buffer such as PBS).Optionally, washing and/or concentrating comprises washing with aculture medium. The culture medium can be any medium suitable forexpansion of MSCs without differentiation (e.g., DMEM; RPMI; etc. in thepresence of FBS or serum-free replacement). Optionally, the culturemedium contains L-alanyl-L-glutamine and FBS, for example, as set forthin Example 5. Optionally, the culture medium comprises DMEM,L-alanyl-L-glutamine, and about 10% FBS. Optionally, the culture mediumcomprises DMEM with about 4 mM L-alanyl-L-glutamine+about 10% FBS.

In one embodiment, nucleated bone marrow cells are washed and/orconcentrated in an automated device, for example, as set forth inExample 5.

In one embodiment, nucleated bone marrow cells are washed orconcentrated (or washed and concentrated) in a device that allows for afunctionally closed system of filling, sampling, or transferring ofsolutions, for example, as set forth in Example 5.

Establishing an MSC Culture

An MSC culture can be established from MSC-containing tissue or anMSC-containing population (e.g., isolated nucleated bone marrow cells)by any method known in the art.

In one embodiment, an MSC culture is provided by establishing anadherent culture (e.g., a primary adherent culture). Methods ofestablishing an adherent MCS culture are known in the art. Generally,establishing an adherent culture comprises seeding an MSC-containingpopulation (e.g., isolated nucleated bone marrow cells or INBMCs) on asubstrate, as is known in the art (e.g., a treated or untreated plasticculture vessel), and incubating the population under appropriateconditions for expansion of adherent cells. Non-adherent cells areremoved by changing the culture medium with an optional washing step.

Optionally, the culture vessel comprises a cell factory (CF).Optionally, seeding an MSC-containing population comprises seeding at atarget concentration, for example, as set forth above or in Example 6.

Optionally, seeding comprises providing an MSC-containing population insuspension at a target concentration in a container and transferring thesuspension to a culture vessel (e.g., a ten-stack CF), for example, asset forth in Example 6.

Optionally, seeding is performed in an automated device, for example, asset forth in Example 6. Optionally, seeding is performed in a devicethat allows for a functionally closed system of filling, sampling, andtransferring of solutions, for example, as set forth in Example 6.

Cultured MSCs are is incubated under appropriate conditions, as is knownin the art (e.g., at a desired temperature, CO₂ concentration, andrelative humidity), for example, as set forth in Example 6.

Expansion in Culture

Once a culture of MCSs has been established (e.g., by the methodsdescribed above), the culture is expanded to increase the quantity ofMCSs.

The culture is expanded in any culture medium suitable for expansion ofMSCs without differentiation (e.g., DMEM, RPMI, etc. in the presence ofFBS or serum-free replacement). Optionally, the culture medium containsL-alanyl-L-glutamine and FBS. Optionally, the culture medium comprisesDMEM, L-alanyl-L-glutamine, and about 10% FBS. Optionally, the culturemedium comprises DMEM with about 4 mM L-alanyl-L-glutamine and about 10%FBS.

While growing in culture, cells consume nutrients in the culture medium.Optionally, nutrients are replenished by adding culture medium.Optionally, replenishing comprises one or more feed changes (e.g., aboutevery 3-4 days). Optionally, a feed change comprises removing spentmedium (medium with reduced nutrient levels) and then adding freshculture medium. Optionally, non-adherent cells are removed with thespent medium (e.g., by aspiration) during the one or more feed changes,for example, as set forth in Example 7. Optionally, the cells are grownin culture for about 14±2 days (e.g., between passages). Optionally,cells are grown in culture for about 9 to about 12 days for the finalexpansion stage, for example, to produce a P5 culture. Optionally, MSCsare seeded at about 5,900 cells±about 5%, ±about 10%, or ±about 20% percm² or in a range of about 1,000 to about 10,000 and/or about37.5×10⁶±about 5%, ±about 10%, or ±about 20% cells per about 1.5 L ofculture media for expansion steps (e.g., prior to a second or laterexpansion after an initial passage of an MSC culture expanded from apopulation of INBMCs).

MSCs in culture will generally continue growing until confluence atwhich time contact inhibition causes cessation of cell division orgrowth. A passage can then be performed at or before confluence, asdescribed below, to reseed the culture at a reduced concentration inorder to continue expansion of the cells (“passage expansion”), asdescribed above.

Passaging

The methods of manufacture of the present invention comprise at leastone passaging, splitting or “subculturing”.

In one embodiment, one passage comprises removing non-adherent cells,leaving adherent MSCs. Such MSCs can then be dissociated from thesubstrate or flask (e.g., by using a protease such as trypsin orcollagenase), media can be added, optional washing (e.g., bycentrifugation) may be performed, and then the MSCs can be replated orreseeded to one or more culture vessels contain a greater surface areain total, for example, as set forth in Example 9 through Example 11.Other methods of removing non-adherent cells include steps ofnon-enzymatic treatment (e.g., with EDTA).

The cells can then continue to expand in culture, as described above.

Optionally, cells are passaged at or near confluence (e.g., about 75% toabout 95% confluence), for example, as set forth in Example 8, Example17, Example 18, or Example 20.

In one embodiment, the MSC preparations in culture are split at a ratioof about 1:6 (by surface area). Optionally, the MCSs in culture aresplit at a ratio of about 1:2 or more, about 1:3 or more, about 1:4 ormore, about 1:5 or more, about 1:6 or more, about 1:7 or more, about 1:8or more, about 1:9 or more, or about 1:10 or more.

In one embodiment, the MSCs are seeded at a concentration of about37.5×10⁶±about 5%, about 10%, about 15%, or about 20% cells/ml ofculture medium.

In one embodiment, the MSCs are cultured for at least about twopassages. Optionally the MSCs are cultured for about two to about five,two to about six, or two to about seven passages, or more. Optionally,the MSCs are cultured for about two, about three, about four, or aboutfive passages. Optionally, the MSCs are passaged not more than about tenor not more than about seven passages.

Cell Handling

The process of MSC isolation and ex-vivo expansion can be performedusing any equipment and cell handing methods known in the art.

Various manufacturing embodiments of the present invention employ stepsthat require manipulation of cells, for example, steps of seeding,feeding, dissociating an adherent culture, or washing. Any step ofmanipulating cells has the potential to insult the cells. Although MSCscan generally withstand a certain amount of insult during preparation,cells are preferably manipulated by handling procedures and/or equipmentthat adequately performs the given step(s) while minimizing insult tothe cells.

Steps of washing and freezing are potentially insulting to cells.Without being bound by theory, it is believed that protocols requiringextensive washing or rapid freezing of cells do not provide the qualityor consistency required to manufacture a pharmaceutical compositionsuitable for administration to a human. Furthermore, the effects ofextensive washing or rapid freezing protocols on the viability of cellsand the efficacy a pharmaceutical composition comprising such cells isunknown.

In one embodiment, the mesenchymal stem cells are washed in an apparatusthat includes a cell source bag, a wash solution bag, a recirculationwash bag, a spinning membrane filter having inlet and outlet ports, afiltrate bag, a mixing zone, an end product bag for the washed cells,and appropriate tubing, for example, as described in U.S. Pat. No.6,251,295 to Johnson, which is hereby incorporated by reference. Theapparatus can be a closed or semi-closed system, thereby reducing thepotential for contamination. Unwashed MSCs from the cell source bag canbe mixed with the wash solution in the centrifugal filtration device.The resulting suspension of mesenchymal stem cells in wash solution thenis fed to the spinning membrane filter through an inlet port. A filtratecomprising wash solution is withdrawn from the spinning membrane filterthrough a first outlet port, and a concentrated suspension of MSCs iswithdrawn from the spinning membrane filter through a second outletport, and fed into the recirculation wash bag. The MSCs then arewithdrawn from the recirculation wash bag, mixed with additional washsolution, and sent again to the spinning membrane filter.

In one embodiment, washing comprises pooling several culture vessels ina single container and washing, for example, as set forth in Example 12and Example 28.

In one embodiment of the present technology, a therapeutic MSCpreparation is provided by placing culture expanded MSCs in anappropriate cryopreservative for cryopreservation. Optionally, thecryopreservative comprises DMSO. Optionally, an MSC preparationcomprises about 5% to about 20% cryopreservative. For example, in someembodiments, a therapeutic MSC preparation comprises MSCs and about 20%DMSO. In other embodiments, a therapeutic MSC preparation comprises MSCsand about 10% DMSO. In some embodiments, the DMSO is added to purifiedMSCs.

In one embodiment, cells are cryogenically frozen and/or stored at lessthan about ≤−70° C., for example: about ≤−80° C., about ≤−80° C. toabout ≤−135° C., or about ≤−135° C. In one embodiment, cells arecryogenically frozen by reducing the preparation temperature by aboutone degree per minute. Optionally, cells are cryogenically frozen byreducing the preparation temperature by about one degree per minuteuntil the preparation reaches about ≤−80° C., and then the temperatureof preparation is reduced to about ≤−135° C. Optionally, the rate oftemperature change before reaching about −80° C. is less than the rateof temperature change from about ≤−80° C. to about ≤−135° C. Optionally,the temperature of preparation is reduced to about ≤−80° C. in a firstrefrigerating apparatus that provides a controlled rate of temperaturechange (e.g., a Cryomed) while temperature of preparation is reducedfrom about ≤−80° C. to about ≤−135° C. in second refrigerating apparatusthat does not provide a controlled rate of temperature change, forexample, a freezer set at about −135° C. (e.g., a LN2 freezer), forexample, as discussed in Example 29.

In one embodiment, manufacturing of MSCs preparation is performed in aclosed or semi-closed system, for example, for filling, sampling, andtransfer of solutions. A closed or semi-closed system can be provided byutilizing transfer means (e.g., tubes) which are sterile-welded,sterile-fused, sterile-sealed, or otherwise sterilely connected tocell-containing vessels. Examplary steps that can use the welder andsealer include: transfer of BMA and fluids, sampling, seeding of CF,feeds, passages, harvest, formulation, fill and cryopreservation.Examplary cell-containing vessels include those which are used in thesteps described above, for example, BMA collection containers or bags,cell washing containers or bags, cell-cryopreservation vessels or bags,cell-culture vessels, and cell-sampling vessels.

Screening

In one embodiment of the present invention, an MSC preparation isscreened for therapeutic use, wherein the preparation is selected foradministration to a patient if the preparation has an antigen profiletaught herein (e.g., as set forth in Example 30 or Example 32).Optionally, the preparation is selected for administration only if ithas an acceptable potency (e.g., a TNFRI profile set forth in Example 30or Example 32), an acceptable post-thaw viability (e.g., as set forth inExample 30 or Example 32), has an acceptable differentiation profile(e.g., as set forth in Example 30 or Example 32), has a pathogen contentunder an acceptable limit, and/or has an animal-derived residual contentunder an acceptable limit.

A number of methods are known in the art for determining an antigenprofile. In one embodiment, an antigen profile is determined by flowcytometry or fluorescence activated cell sorting (FACS). Thus, as knownin the art, FACS involves exposing cells to a reporter, such as alabeled antibody, which binds to and labels antigens expressed by thecell. Methods of production of antibodies and labeling thereof to formreporters are known in the art. The cells are then passed through a FACSmachine, which sorts the cells from each other based on the labeling.

The potency in the TNFRI profiles of the present invention is therelative amount of TNFRI in the preparation (pg per million MSCs) andoptionally, the activity of the preparation (% IL-2Rα Inhibition). Theamount of cellular TNFRI in a culture of mesenchymal stem cells can bedetermined by methods known to those skilled in the art. Such methodsinclude, but are not limited to, quantitative assays such asquantitative ELISA assays, for example, as described in WO 2007/087139to Danilkovitch et al., which is hereby incorporated by reference. It isto be understood, however, that the scope of the present invention isnot to be limited to any particular method for determining the amount.The TNFRI activity of the preparation is expressed as the percent IL-2Rαexpression inhibition on mitogen-stimulated white blood cells and isdetermined by the method described by Le Blanc et al. (“Mesenchymal StemCells Inhibit the Expression of CD25 (Interleukin-2 Receptor) and CD38on Phytohaemagglutinin-Activated Lymphocytes”, Scandinavian Journal ofImmunology, 2004, Vol 60 No 3, Pages 307-315).

Methods of determining adipogenic, chondrogenic, and osteogenicdifferentiation are described by Pittenger et al. (“Multilineagepotential of adult human mesenchymal stem cells”. Science 1999;284:143-7). Adipogenic differentiation can be measured by detecting theformation of lipid-rich vacuoles within cells after treatment with1-methyl-3-isobutylxanthine, dexamethasone, insulin, and indomethacin.Chondrogenic differentiation can be measured by detecting type IIcollagen and expression of chondrocytic markers after culturing thecells without serum and with transforming growth factor-b3. Osteogenicdifferentiation is measured by detecting alkaline phosophataseexpression after treatment with dexamethasone, ß-glycerol phosphate, andascorbate and in the presence of about 10% v/v FBS.

In one embodiment of the present invention, an MSC preparation isscreened for therapeutic use, wherein the preparation is selected foradministration to a patient if the preparation is free of pathogens.Optionally, the pathogens screened for include on or more of endotoxin,bacteria, fungi, or viruses. Optionally, the viruses include one or moreof a human T-cell lymphotropic virus nucleic acid sequence, a humanhepatitis virus nucleic acid sequence, a human cytomegalovirus nucleicacid sequence, a human Epstein-Barr virus nucleic acid sequence, a humanherpesvirus nucleic acid sequence, a human immunodeficiency virusnucleic acid sequence, a parvovirus nucleic acid sequence, and a humanpapillomavirus nucleic acid sequence. Methods for screening for suchpathogens are well known in the art.

In one embodiment of the present invention, an MSC preparation isscreened for therapeutic use, wherein the preparation is selected foradministration to a patient if the preparation has a BSA and/or trypsincontent of less than a predetermined amount. Optionally, a presentpreparation contains less than about 55 μg/mL, about 42 μg/mL, about 25μg/mL, about 13 μg/mL, about 10 μg/mL BSA, about 7 μg/mL, or about 15μg/mL BSA. Optionally, a present preparation contains less than: about42 μg/mL or about 40 μg/mL trypsin. Such examplary methods includequantitative assays such as quantitative immuno assays, for example, asdescribed in copending application Ser. No. 12/541,282, filed Aug. 14,2009, which is hereby incorporated by reference.

In one embodiment of the present invention, an MSC preparation isscreened for therapeutic use, wherein the preparation is selected foradministration to a patient if the preparation has acceptablefreeze-thaw viability (e.g., as set forth in Example 30 or Example 32).Cell viability and cell count is assessed by flow cytometry offluorescently stained cells, based on differential staining of viableand non-viable cells due to their respective permeabilities toDNA-binding dyes in a proprietary reagent (Viacoun™, GuavaTechnologies). The stained sample is analyzed on a Guava PCA flowcytometer and the Guava CytoSoft software package. The Guava PCA uses a532 nm diode laser to excite the dyes, whose fluorescence is detected byphotomultipliers (“PM”)(PM1 detects the viability dye emission at 580nm; PM2 detects nuclear dye emission at 675 nm), while a photodiodemeasures forward light scatter (FSC). The fluorescence signal isresolved, allowing quantitation of the viable and non-viable cellpopulations from cellular debris in the test article. The Guava'soperational range of diluted cell suspension is about 10 to about 500particles/mL.

Methods of Use

In one embodiment, the invention provides a method of treating a subjectin need thereof comprising the step of administering a therapeutic doseof an MSC preparation having an antigen profile taught herein (e.g., asset forth in Example 30 and Example 32). Optionally, the preparationfurther comprises a second profile taught herein (e.g., as set forth inExample 30 or Example 32).

In one embodiment, the disease is an autoimmune disease or agraft-versus-host disease treated by the administration of one or moretherapeutic doses. Optionally, administration of one or more therapeuticdoses produces an endpoint taught herein (e.g. overall response orpartial response, such as that of a single organ). Optionally,administration of one or more therapeutic doses is therapeuticallyeffective in steroid-refractory disease (e.g., GVHD).

By way of example, autoimmune diseases which can be treated inaccordance with the present invention include multiple sclerosis, Type 1diabetes, rheumatoid arthritis, uveitis, autoimmune thyroid disease,inflammatory bowel disease (IBD), scleroderma, Graves' Disease, lupus,Crohn's disease, autoimmune lymphoproliferative disease (ALPS),demyelinating disease, autoimmune encephalomyelitis, autoimmunegastritis (AIG), and autoimmune glomerular diseases. Also, as notedhereinabove, graft-versus-host disease (GVHD) can be treated. It is tobe understood, however, that the scope of the present invention is notto be limited to the treatment of the specific diseases mentionedherein.

In general, the mesenchymal stem cell therapy is based, for example, onthe following sequence: harvest of MSC-containing tissue, isolate andexpand MSCs, and administer the MSCs to the animal, with or withoutbiochemical or genetic manipulation.

A therapeutic dose for an autoimmune disease or graft-versus-hostdisease can contain about 1×10⁵ cells/kg to about 1×10⁷ cells/kg. Inanother embodiment, a therapeutic dose is about 1×10⁶ cells/kg to about5×10⁶ cells/kg. In another embodiment, a therapeutic dose is about 2×10⁶cells/kg to about 8×10⁶ cells/kg. In another embodiment, a therapeuticdose is about 2×10⁶ cells/kg or about 2×10⁶±about 10%, about 20%, orabout 30% cells/kg. In another embodiment, a therapeutic dose is about8×10⁶ cells/kg or about 8×10⁶±about 10%, about 20%, or about 30%cells/kg, and include any amounts or ranges there between. Given an MSCpreparation of the present invention, the amount of mesenchymal stemcells to be administered is dependent upon a variety of factors,including the age, weight, and sex of the patient, the autoimmunedisease to be treated, and the extent and severity thereof.

The mesenchymal stem cells can be administered by a variety ofprocedures. The mesenchymal stem cells can be administered systemically,such as by intravenous, intraarterial, or intraperitonealadministration. The mesenchymal stem cells can be administered by directinjection to an organ or tissue in need thereof. The mesenchymal stemcells can be applied topically. The mesenchymal stem cells can beapplied directly to a tissue in need thereof during a surgicalprocedure.

The mesenchymal stem cells can be, for example, autologous, allogeneic,or xenogeneic.

The mesenchymal stem cells can be administered in conjunction with anacceptable pharmaceutical carrier. For example, the mesenchymal stemcells can be administered as a cell suspension in a pharmaceuticallyacceptable liquid medium or gel for injection or topical application.

In accordance with another aspect of the present invention, there isprovided a method of treating an inflammatory response in an animal. Themethod comprises administering to the animal mesenchymal stem cells inan amount effective to treat the inflammatory response in the animal.

Although the scope of this aspect of the present invention is not to belimited to any theoretical reasoning, it is believed that themesenchymal stem cells promote T-cell maturation to regulatory T-cells(TReg), thereby controlling inflammatory responses. It is also believedthat the mesenchymal stem cells inhibit T helper 1 cells (Th1 cells),thereby decreasing the expression of the Interferon-γ (IFN-γ) in certainTh1 type inflammatory reactions, such as those associated withpsoriasis, for example.

In one embodiment, the inflammatory responses which can be treated arethose associated with Th1-type diseases.

In one embodiment, the inflammatory responses which can be treated arethose associated with hyperactivated T helper 2 cells (Th2 cells), forexample, allergies and asthma.

In one embodiment, the inflammatory responses which can be treated arethose associated with psoriasis.

In another embodiment, the mesenchymal stem cells can be administered toan animal such that the mesenchymal stem cells contact microglia orastrocytes in the brain to reduce inflammation. Although this embodimentis not to be limited to any theoretical reasoning, it is believed thatthe mesenchymal stem cells limit neurodegeneration caused by activatedglial cells in diseases or disorders such as Alzheimer's Disease,Parkinson's Disease, stroke, or brain cell injuries.

In yet another embodiment, the mesenchymal stem cells can beadministered to an animal such that the mesenchymal stem cells contactkeratinocytes and Langerhans cells in the epidermis of the skin toreduce inflammation as can occur in psoriasis, chronic dermatitis, andcontact dermatitis. Although this embodiment is not to be limited to anytheoretical reasoning, it is believed that the mesenchymal stem cellscan contact the keratinocytes and Langerhans cells in the epidermis, andalter the expression of T-cell receptors and cytokine secretion profilesleading to decreased expression of tumor necrosis factor-alpha (TNF-α)and increased regulatory T-cell (Treg cell) population.

In a further embodiment, the mesenchymal stem cells can be used toreduce inflammation in an articulating joint, the bone, cartilage, orcartilage and bone, as occurs in arthritis and arthritis-likeconditions, including but not limited to, osteoarthritis and rheumatoidarthritis, and other arthritic diseases listed in the websitewww.arthritis.org/conditions/diseases as it appears on 12 September2011. Although the scope of this embodiment is not intended to belimited to any theoretical reasoning, it is believed that themesenchymal stem cells can inhibit Interleukin-17 secretion by memoryT-cells in the synovial fluid.

In another embodiment, the mesenchymal stem cells can be used to limitinflammation in the gut and liver during inflammatory bowel disease andchronic hepatitis, respectively. Although the scope of this aspect ofthe present invention is not intended to be limited to any theoreticalreasoning, it is believed that the mesenchymal stem cells promoteincreased secretion of Interleukin-10 (IL-10) and the generation ofregulatory T-cells (Treg cells).

In another embodiment, the mesenchymal stem cells can be used to inhibitexcessive neutrophil and macrophage activation in pathologicalconditions such as sepsis and trauma, including burn injury, surgery,and transplants. Although the scope of this embodiment is not to belimited to any theoretical reasoning, it is believed the mesenchymalstem cells promote secretion of suppressive cytokines such as IL-10, andinhibit IL-1 RA.

In another embodiment, the mesenchymal stem cells can be used to controlinflammation in immune privileged sites such as the eye, including thecornea, lens, pigment epithelium, and retina, brain, spinal cord,pregnant uterus and placenta, ovary, testes, adrenal cortex, liver, andhair follicles. Although the scope of this embodiment is not to belimited to any theoretical reasoning, it is believed that themesenchymal stem cells promote the secretion of suppressive cytokinessuch as IL-10 and the generation of Treg cells.

In yet another embodiment, the mesenchymal stem cells can be used totreat tissue damage associated with end-stage renal disease (ESRD)during dialysis or glomerulonephritis. Although the scope of thisembodiment is not to be limited to any theoretical reasoning, it isbelieved that mesenchymal stem cells can promote renal repair.Mesenchymal stem cells also are believed to express and secrete vascularendothelial growth factor, or VEGF, which stimulates new blood vesselformation, which may aid in the repair of damaged kidney tissue.

In a further embodiment, the mesenchymal stem cells can be used tocontrol viral infections such as influenza, hepatitis C, Herpes SimplexVirus, vaccinia virus infections, and Epstein-Barr virus. Although thescope of this embodiment is not to be limited to any theoreticalreasoning, it is believed that the mesenchymal stem cells promote thesecretion of Interferons including Interferon-Alpha, Interferon-Beta,and Interferon-Gamma.

In yet another embodiment, the mesenchymal stem cells can be used tocontrol parasitic infections such as Leishmania infections andHelicobacter infections. Although the scope of this embodiment is not tobe limited to any theoretical reasoning, it is believed that themesenchymal stem cells mediate responses by T helper 2 (Th2) cells, andthereby promote increased production of Immunoglobulin E (IgE) byB-cells.

In another embodiment, the mesenchymal stem cells can be administered toan animal to treat inflammation which results from a lung disease ordisorder. Such lung diseases or disorders include, but are not limitedto, Acute Respiratory Distress Syndrome (ARDS), Chronic ObstructivePulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), asthma,and pulmonary hypertension.

Although the scope of this embodiment is not to be limited to anytheoretical reasoning, the inflammatory response in the above-mentionedlung diseases or disorders involves the secretion of TNF-alpha and/orMCP-I. It is believed that the mesenchymal stem cells migrate toinflamed lung tissue due to increased production of one or both ofTNF-alpha and MCP-I, which are chemoattractants for mesenchymal stemcells.

It is to be understood, however, that the scope of this aspect of thepresent invention is not to be limited to the treatment of anyparticular inflammatory response.

The mesenchymal stem cells can be administered to a mammal, includinghuman and non-human primates, as hereinabove described.

The mesenchymal stem cells also can be administered systemically, ashereinabove described, for example, but not limited to, intravenously orintra-arterially. In some embodiments, the MSCs of the presenttechnology can be administered by direct injection into the tissue, forexample, but not limited to, direct injection to inflamed tissue in asubject or a joint of a subject. For example, MSCs of the presenttechnology can be administered directly to the heart of a patient.Alternatively, in the case of osteoarthritis or rheumatoid arthritis,the mesenchymal stem cells may be administered directly to an arthriticjoint.

The mesenchymal stem cells, in accordance with the present invention,can be employed in the treatment, alleviation, or prevention of anydisease or disorder which can be alleviated, treated, or preventedthrough angiogenesis. Thus, for example, the mesenchymal stem cells canbe administered to an animal to treat blocked arteries, including thosein the extremities, i.e., arms, legs, hands, and feet, as well as theneck or in various organs. For example, the mesenchymal stem cells canbe used to treat blocked arteries which supply the brain, therebytreating or preventing stroke. Also, the mesenchymal stem cells can beused to treat blood vessels in embryonic and postnatal corneas and canbe used to provide glomerular structuring. In another embodiment, themesenchymal stem cells can be employed in the treatment of wounds, bothinternal and external, as well as the treatment of dermal ulcers foundin the feet, hands, legs or arms, including, but not limited to, dermalulcers caused by diseases such as diabetes and sickle cell anemia.

Furthermore, because angiogenesis is involved in embryo implantation andplacenta formation, the mesenchymal stem cells can be employed topromote embryo implantation and prevent miscarriage.

In addition, the mesenchymal stem cells can be administered to an unbornanimal, including humans, to promote the development of the vasculaturein the unborn animal.

In another embodiment, the mesenchymal stem cells can be administered toan animal, born or unborn, in order to promote cartilage resorption andbone formation, as well as promote correct growth plate morphogenesis.

The mesenchymal stem cells can be genetically engineered with one ormore polynucleotides encoding a therapeutic agent. The polynucleotidescan be delivered to the mesenchymal stem cells via an appropriateexpression vehicle. Expression vehicles which can be employed togenetically engineer the mesenchymal stem cells include, but are notlimited to, retroviral vectors, adenoviral vectors, and adeno-associatedvirus vectors.

The presently described technology and its advantages will be betterunderstood by reference to the following examples. These examples areprovided to describe specific embodiments of the present technology. Byproviding these specific examples, it is not intended limit the scopeand spirit of the present technology. It will be understood by thoseskilled in the art that the full scope of the presently describedtechnology encompasses the subject matter defined by the claimsappending this specification, and any alterations, modifications, orequivalents of those claims.

EXAMPLES Example 1 Donor Selection

The starting material for the production of the present MSCs in thisexample is a bone marrow aspirate (“BMA”) obtained from a human donor.The donor is selected based on the results of one or more or allassessments/tests listed in Table 2.

TABLE 2 Donor Selection Assessment/Test Acceptance Criteria ClinicalBone Marrow Donor Must be Completed Application Consent Form for theBone Must Consent Marrow Collection Informed Consent & Agreement MustConsent for HIV Testing Health & TSE-Risk Factor As accepted by MedicalQuestionnaire (TSE = transmissible Director spongiform encephalopathy)Physical assessment As accepted by Medical Director ABO Rh Results asreported HLA Typing Results as reported HLA Beta DR Typing Results asreported Comprehensive Metabolic Panel Within acceptable limits (CMP)Complete Blood Count (CBC) Within acceptable limits

The qualified donor meets all of the following selection criteria: aminimum of one successful donation, a previous bone marrow cell countgreater than 2.0×10⁶ cells/mL, a history of negative infectious diseasetests, a Body Mass Index (BMI)<30, and an age of 18 to 30 years.

Example 2 Donor Screening

The BMA donor is screened for acceptance by testing a sample of bloodagainst a panel of infectious diseases as shown in Table 3. The donormeets all of the acceptance criteria shown in Table 3.

TABLE 3 Donor Screening Acceptance Regulatory Disease Assay/TestCriteria Status Human Genetic Systems Negative or FDA ImmunodeficiencyHIV-1/HIV-2 Plus 0 Nonreactive Licensed Viruses 1 and 2 EIA (Bio-Rad fordonor (HIV) Antibody Laboratories, screening HIV-1, HIV-2 Hercules, CA)HIV-1. HIV-1 NAT Procleix HIV- Negative or FDA 1/HCV Assay NonreactiveLicensed (Gene-Probe, Inc, for donor San Diego, CA) screening HepatitisB Virus Genetic System Negative or FDA (HBV). Hep B HBsAg EIA 3.0Nonreactive Licensed surface Antigen (Bio-Rad for donor (HBsAg)Laboratories, screening Hercules, CA) Hepatitis B Virus Ortho HBc ELISANegative or FDA (HBV). Antibody Test (Ortho-Clinical NonreactiveLicensed Hep B core (total) Diagnostics, for donor Rochester, NY)screening Hepatitis B Virus COBAS Negative or FDA (HBV). HBV NATAmpliScreen HBV Nonreactive Licensed Test (Roche for donor MolecularSystems screening Inc, Pleasanton, CA) Hepatitis C Virus Ortho HCVVersion Negative or FDA (HCV). 3.0 ELISA Test Nonreactive LicensedAntibody HCV System (Ortho- for donor Clinical Diagnostics, screeningRochester, NY) Hepatitis C Virus Procleix HIV- Negative or FDA (HCV).HCV NAT 1/HCV Assay (Gen- Nonreactive Licensed Probe, Inc., San fordonor Diego, CA) screening Human T Cell Abbott HTLV Negative or FDALymphotropic Virus I/HTLV-II EIA Nonreactive Licensed Types I & II (HTLV(Abbott for donor I1/II). Laboratories, screening Antibody HTLV-I/IIAbbott Park, IL) Cytomegalovirus Capture- Negative or FDA (CMV).Antibody CMV Immucor Nonreactive Cleared CMV Total Gamma (Immucor, fordonor Inc., Norcroos, GA) screening Antibody Epstein- AtheNa Multi-LyteNegative or Laboratory Barr Virus (EBV) EBV (Inverness NonreactiveValidated IgM Medical Professional Diagnostics, Princeton, NJ) RapidProtein Macro-Vue RPR Nonreactive FDA Reagin (RPR, Card Tests (BD, orReactive Cleared for Serological test Franklin Lakes, NJ) Diagnostic forSyphilis) Use Syphilis (Treponema MarDx FTA-ABS Negative or FDApallidum). FTA- Test System (MarDx Nonreactive Cleared ABS performed ifDiagnostics,Inc. for donor RPR is reactive A Trinity Biotech screeningCompany, Carlesbad, CA) West Nile Virus Procleix WNV Negative or FDA(WNV). WNV NAT Assay (Novartis, Nonreactive Licensed Cambridge MA) fordonor screening

Example 3 BMA Collection

Collection of the BMA takes place at an outpatient surgical center(e.g., 7 days after blood sample collection was performed.) The donor isplaced in the prone position and the bone marrow aspiration needle isinserted into the posterior iliac crest. The BMA collection procedureuses two syringes each containing 5 mL of 1,000 USP units/mL heparinsodium, which acts as an anticoagulant. As a result, the BMA materialcontains a small concentration (10,000 U/BMA) of heparin sodium. Up to60 ml bone marrow is aspirated from the insertion site (from each sideof the iliac crest), for example from 100 ml to 120 ml bone marrow intotal.

Example 4 BMA Packaging

The BMA material is packaged in a 300 mL Baxter LifeCell™ tissue culturebag, which is an FDA-cleared, sterile, gas-permeable bag that isintended for the cultivation of cells grown in suspension. The containerhas a tubing harness allowing for functionally closed system filling,sampling, and transferring of solutions during the manufacturingprocess.

Example 5 Isolation of Nucleated Bone Marrow Cells from BMA

The first step (day 1) in the isolation and expansion of humanmesenchymal stem cells (hMSCs) involves the isolation of nucleated bonemarrow cells from the BMA. A CytoMate® Cell Washer (Baxter HealthcareCorp., Deerfield, Ill.) connected with a fluid transfer set is used totransfer a Plasma-Lyte®A (Baxter, Deerfield, Ill.) and Hespanformulation to the BMA by using a Terumo sterile tube welder to fuse theBMA bag tubing lines together with the fluid transfer set. In someembodiments of the present application, other cell washing orpurification machines or techniques are used instead of a Cytomate CellWasher; when the Cytomate is referred to herein, the present inventorsappreciate that any suitable replacement device for cell washing orpurification machines or techniques may be employed instead. Hespan isutilized to agglutinate, sediment, and separate the majority of the redblood cells (RBCs) from the bone marrow nucleated cells. Using the“Fluid Transfer” program on the CytoMate® (Baxter, Deerfield, Ill.), thePlasma-LyteA (Baxter, Deerfield, Ill.) and Hespan formulation (6%Hetastarch) is then transferred into the BMA bag and the RBCs areallowed to settle for approximately 60 to 90 minutes until a distinctseparation appears between the nucleated bone marrow cells and the RBCs.The nucleated bone marrow cells (top layer) are isolated from the BMAbag using a Fenwal plasma extractor to press the upper nucleated bonemarrow cell layer into a transfer pack. The isolated nucleated bonemarrow cells (INBMCs) are then transferred to the CytoMate® andprocessed by concentrating and washing the cells with culture medium(DMEM w/4 mM L-alanyl-L-glutamine+10% FBS). INBMCs are counted using aHematology Analyzer (Beckman Coulter).

Example 6 Isolation of MSCs

Following the cell count, the INBMCs are diluted to the target seedingconcentration (e.g., 925×10⁶ INBMCs per 55 ml) and transferred to a 1.5L culture medium bag using the “Transfer Volume” program on theCytoMate® to obtain the “Media Cell Suspension”. The “Media CellSuspension” is used to seed the INBMCs into a CO₂ primed Nunc ten-stackcell factory (CF) at about 5,900 cells±about 20% per cm² of growingsurface. The CF is then placed in an incubator set at about 37±1° C. andabout 5±2% CO₂, and ambient relative humidity. This is the primaryculture (P0). After the initial seeding of the INBMCs, MSCs attach tothe tissue culture plastic and grow to form a primary adherentpopulation.

Example 7 Feeds

Non-adherent cells are aspirated away during a feed change. The feed isan addition of 1.5 L of fresh culture medium to replace the existingculture medium (“spent medium”) that has been depleted of nutrients fromcells growing in culture. A feed is performed every 3-4 days. Duringeach feed, the CF is examined for integrity and appearance.

Example 8 First Passage

After approximately about 21±3 days in culture, the primary culture (P0)is expanded (e.g., from one CF to approximately six CFs, or, optionallyfrom one CF to approximately eight CFs) for the first passage (P1).

Example 9 Trypsinizing

The spent medium in each CF is drained via gravity flow into an emptymedium bag that is attached (e.g., sterile welded such as using a TerumoSterile Tubing Welder) to a CF tubing set. After the CF has beencompletely drained, the “spent medium” bag is removed.

A “Stop Solution” bag and a “Trypsin-EDTA (0.05% Trypsin, 0.53 mM EDTA)”bag are attached (e.g., sterile welded) to the CF tubing set.Trypsin-EDTA approximately 400 mL) is added to the CF via gravity flow.Once the “Trypsin-EDTA” bag has been emptied, the CF is placed into a37±1° C. and 5±2% CO₂ incubator for trypsinization.

Each CF is trypsinized for up to 30 minutes (e.g., up to about 15 toabout 30 min or any range in-between). During the trypsinization period,the CFs are observed approximately every 8 minutes using an invertedmicroscope to determine the percentage of cells that have detached. Whenthe percentage of detached cells is estimated to be >about 90%, thetrypsinization is stopped by adding 100 mL of media “stop solution”(e.g., DMEM containing 10% FBS) into the CF via gravity flow. Theduration of the trypsinization is recorded.

Example 10 Washing

The trypsinized-stopped cell suspension is drained via gravity flow intoa 600 mL transfer pack that is attached to a CF tubing set.

The trypsinized-stopped cell suspension is then washed using theCytoMate®.

Example 11 Seeding

The hMSCs are counted on a Guava Personal Cytometer™ (GuavaTechnologies).

An amount of cells (e.g., about 37.5×10⁶ cells or about 37.5×10⁶±about5%, about 10%, or about 20% cells) are added to 1.5 L culture mediumbags on the CytoMate®. The culture medium bags now containing cells arethen used to seed a corresponding number of CFs via sterile tubingconnections (e.g. at about 5,900 cells/per cm²±about 5%, about 10%,about 15%, or about 20% per cm²). The seeded CFs are then placed in anincubator set at about 37±1° C. and about 5±2% CO₂, and ambient relativehumidity for approximately 14±2 days, with feeds approximately every 3-4days, as set forth in Example 7.

Example 12 Second Passage

After approximately 14±2 days in culture, the CF (e.g., six-CF, oreight-CF) P1 cultures are expanded (e.g., into 36 CFs) for the secondpassage (P2).

Each of the CFs (e.g., 6 CFs, or eight-CF) is passaged as set forth inExample 9 through Example 11 (e.g., to provide 36 CFs). Thetrypsinized-stopped cell suspensions of several CFs can be pooled beforewashing.

Example 13 Harvest

After approximately 14±2 days in culture, the cultured hMSCs areharvested (e.g., after about 42 to about 56 days total over twopassages). Each CF is processed as set forth in Example 9 throughExample 10, using Plasma-Lyte® A containing about 1% Human Serum Albuminas a stop solution. The trypsinized-stopped cell suspensions of severalCFs can be pooled before washing.

Example 14 Preparing an In-Process Intermediate

Based on the cell yield calculated from the harvest, the pooled “WashedhMSCs”, are concentrated or diluted to obtain an in-process intermediateformulation containing about 6.0 million cells/mL, for example, in awash solution such as about 1% HSA in Plasma-Lyte® A. The in-processintermediate is a clinical scale MSC preparation that provides astandardized formulation for sample analysis or cryopreservation beforefurther expansion, if desired, or before administration to a patient astherapy.

Samples for sterility, endotoxin, adventitious viruses (DNA PCR and RNAPCR detection), and phenotype lot release testing are obtained from theformulation.

The in-process intermediate is diluted with an equal volume (1:1) ofcryoprotectant solution containing about 20% DMSO and about 9% HSA inPlasma-Lyte®A. The in-process intermediate has now been diluted to about3.0 million cells/mL in approximately 10% DMSO and approximately 5% HSAin Plasma-Lyte® A.

Samples for potency, cell line species identity, human karyotyping, andultrastructural evaluation of cell culture for viral particles(transmission electron microscopy or “TEM”) for lot release testing arethen obtained from the in-process intermediate.

Example 15 Cryopreservation and Storage

A number of Cryocyte™ (Baxter, Deerfield, Ill.) bags (between about 100to about 300, e.g. about 200) are filled, each with a 15 mL volume ofthe in-process intermediate. Each bag now contains about 45 million MSCsin about 5% HSA, about 10% DMSO in Plasma-Lyte®A (Baxter, Deerfield,Ill.). The in-process intermediate is then placed into a secondarypackaging container (e.g. a Custom Biogenic Systems aluminumcanister/cassette) that is designed to enclose and protect the Cryocyte™bag. Once in the cassette, the in-process intermediate is cryopreservedin a Cryomed controlled-rate freezer. Following completion of thefreezing program, the frozen bags are then transferred from the Cryomed®(Thermo Scientific, Rockford, Ill.) and placed into a LN₂ cryofreezer.

Before freezing, a sample is obtained for sterility, endotoxin,appearance, and cell viability (post-thaw) testing. The in-processintermediate is screened for acceptance by each of the tests listed inTable 4.

TABLE 4 Preparation Screen DS Test Name DS Sample Point DS SpecificationSterility 2X DCB Formulation- Negative (2X DCB Formulation) HarvestMycoplasma PTC 2X DCB Formulation- Negative Harvest Phenotype (FACS) 1XDCB Formulation- ≥95% CD166 CD166 Harvest Phenotype (FACS) 1X DCBFormulation- ≥95% CD105 CD105 Harvest Phenotype (FACS) CD45 1X DCBFormulation- ≤1.25% CD45   Harvest Detection of HCV RNA 2X DCBFormulation- Negative by RT-qPCR Harvest Detection of CMV DNA 2X DCBFormulation- Negative by qPCR Harvest Detection of EBV DNA 2X DCBFormulation- Negative by qPCR Harvest Detection of HBV DNA 2X DCBFormulation- Negative by qPCR Harvest Detection of HHV-6 2X DCBFormulation- Negative Variant A and Variant B Harvest DNA by qPCRDetection of HHV-8 DNA 2X DCB Formulation- Negative by qPCR HarvestDetection of HIV-1 DNA 2X DCB Formulation- Negative by qPCR HarvestDetection of HIV-2 DNA 2X DCB Formulation- Negative by qPCR HarvestDetection of HTLV 1 & II 2X DCB Formulation- Negative DNA by qPCRHarvest Detection of Parvovirus 2X DCB Formulation- Negative B-19 DNA byqPCR Harvest Detection of HPV 18 2X DCB Formulation- Negative DNA byqPCR Harvest In-Vivo Assay for Viral 2X DCB Formulation + NegativeContaminants (USFDA): Spent Medium-Harvest Mouse and Egg In-Vitro Assayfor 2X DCB Formulation + Negative Detection of Adventitious SpentMedium-Harvest Viral Contaminants: MRC-5, VERO, Hs68 Cells Thin SectionElectron 2X DCB Formulation + Negative Microscopy for DetectionCryoprotectant-Harvest of Viral Particles Cell Line Species Identity 2XDCB Formulation + Human Cell Line by Isoenzyme Cryoprotectant-HarvestElectrophoresis Karyology-Cell Culture 2X DCB Formulation + NoChromosomal Cryoprotectant-Harvest Abnormalities Sterility DS PackagedDonor Negative (Packaged Dose) Cell Bank Endotoxin DS Packaged Donor≤1.67 EU/mL (Packaged Dose) Cell Bank Potency (TNF RI) 1X DCBFormulation- ≥39 pg/mL (13 Harvest pg/million cells) Potency (IL-2Rα) 1XDCB Formulation- ≥30% Inhibition Harvest of IL-2Rα Expression AppearanceDS Packaged Donor Pass: Opaque Cell Bank contents, off- white to paleamber in color

Example 16 Thawing of the In-Process Intermediate

The in-process intermediate is selected for further culture expansion.Each of the bags is thawed and processed, alone or simultaneously withthe others, and processed by the steps that follow. The bag is dilutedwith DMEM containing about 10% FBS. A sample is taken from the bag for acell count and cell viability assessment, and then hMSCs are transferredfrom the bag into 1.5 L of culture medium.

Example 17 Third Passage

The Culture Media containing hMSCs are seeded into a CF. The CF is thenplaced in an about 37±1° C. and about 5±2% CO₂ incubator and maintainedwith media feeds, as set forth in Example 7, for approximately 14±2days.

Example 18 Fourth Passage

After approximately 14 days±2, the P3 culture is split (e.g., intoapproximately six CFs, or optionally approximately eight CFs) for thefourth passage (P4).

Example 19 Passaging

Each of the CFs (e.g., six CFs) is passaged as set forth in Example 9through Example 11. The trypsinized-stopped cell suspensions of severalCFs can be pooled before washing.

Example 20 Fifth Passage

After approximately 14±2 days (Day 28) in culture, the P4 cultures(e.g., six cultures) are split (e.g., into 40 CFs) for the fifth passage(P5).

Example 21 Passaging

Each of the CFs is passaged as set forth in Example 9 through Example11, except that the CFs are cultured for 9 to 12 days. Thetrypsinized-stopped cell suspensions of several CFs can be pooled beforewashing.

Example 22 Pre-Harvest

After approximately 9 to approximately 12 days in culture (Day 42), thecultured hMSCs are harvested.

Example 23 Harvesting

A harvest is performed, as set forth in Example 13, except that theharvest is performed after 9 to 12 days, as detailed above.

Example 24 Formulating an MSC Preparation for Therapy or Storage

Based on the cell yield calculated from the harvest, the pooled “WashedhMSCs” are either concentrated or diluted to obtain a formulationcontaining about 16.6 million cells/mL. If the pooled “Washed hMSCs” areconcentrated, the cells are spun down and the excess volume is removed.If the pooled “Washed hMSCs” are diluted, the required volume ofWash/Stop Solution (about 1% HSA in Plasma-Lyte®A) is added to get thedesired concentration. A sample for phenotype and residuals (BSA andtrypsin) testing are obtained from the formulation.

The formulation is then diluted with an equal volume (1:1) ofcryoprotectant solution containing about 20% DMSO and about 9% HSA inPlasma-Lyte®A. The formulation now contains a concentration of about 8.3million cells/mL (in approximately 10% DMSO and approximately 5% HSA inPlasma-Lyte A).

A sample for potency testing is obtained from the diluted formulation.

Example 25 Cryopreserving and Storage

The MSC preparation is then filled into a number of Cryocyte™ bags to avolume of about 15 mL/bag (e.g., about 8,000 bags in total, oroptionally about 10,000 bags in total). Each bag of the MSC preparationhas a final formulation of about 125×10⁶ hMSCs (at least about 100×10⁶viable hMSCs after a freeze-thaw cycle) in Plasma-Lyte®A containing aconcentration of about 5% HSA and about 10% DMSO per bag. Bags of theMSC preparation are then placed into a secondary container (a CustomBiogenic Systems aluminum canister/cassette) that is designed to encloseand protect the Cryocyte™ bag. Once in the cassette, the MSC preparationis cryopreserved in a controlled rate freezer (Cryomed®, ThermalScientific, Rockford, Ill.). Following completion of the freezingprogram, the frozen bags are then transferred from the Cryomed® (ThermalScientific, Rockford, Ill.) and placed into LN₂ cryofreezers. A samplebag is obtained for sterility, endotoxin, appearance and cell viability(post-thaw) lot release testing.

Example 26 MSC Preparation Acceptance for Therapy

The MSC preparation remains in “quarantine” pending testing and release.The MSC preparation passes each of the tests listed in Table 5.

TABLE 5 Preparation Screen Test Name Sample Point DescriptionSpecification Sterility Pooled Direct Negative (Pooled SuspensionInoculation Suspension) Bulk-Harvest Method Mycoplasma Pooled Points toNegative (PTC) Suspension Consider Bulk-Harvest Phenotype DP Flow ≥95%CD166 (FACS) Formulation- Cytometry CD166 Harvest Phenotype DP Flow ≥95%CD105 (FACS) Formulation- Cytometry CD105 Harvest Phenotype DP Flow≤0.75% CD45   (FACS) CD45 Formulation- Cytometry Harvest Sterility DPDirect Negative (Packaged Inoculation Dose) Method Endotoxin DPChromogenic ≤1.67 EU/mL (Packaged Kinetic Dose) Method Viability DP Flow≥70% (Post Thaw) Cytometry Residual BSA 2X DP ELISA ≤10 μg/mLFormulation Residual 2X DP ELISA ≤30 μg/mL Trypsin Formulation Potency(TNF DP ELISA Min: ≥108 pg/mL RI) Formulation- (13 pg/million cells)Harvest Max: ≤368 pg/mL (44 pg/million cells) Potency (IL- DP ELISA ≥30%Inhibition of 2Rα) Formulation- IL-2Rα expression Harvest Appearance DPMacroscopic Pass: Opaque Assessment contents, off-white to pale amber incolor, no particulates/fibers, no cell clumping observed, packageintegrity must be intact.

Example 27 Evaluation of Passage Number

The number of cells harvested per donor is correlated with the number ofpassages during culture expansion. Although cells from any passage canbe formulated for therapy, it has been surprisingly discovered thatlimiting the number of passages, for example, to less than any of about14, about 10, about 8, or about 6 passages, provides preparations with asuperior safety profile. By the end of the process set forth in Example1 through Example 23, there are approximately 22 to approximately 25(e.g., 25) population doublings. Without being bound by theory, it isbelieved that limiting the number of passages minimizes the risk ofspontaneous genetic transformations.

Example 28 Evaluation of Washing Protocol

Based on low post-thaw cell viabilities observed in a development study,changes were made in the CytoMate® (Baxter, Deerfield, Ill.) cellwashing process to improve cell viability and cell recovery post-thaw.The old standard procedure to be improved upon here used a CytoMate®(Baxter, Deerfield, Ill.) programmed to wash all three collection bagsserially. The time required to complete the entire wash cycle was threetimes longer than the time required to wash the contents of oneindividual collection bag. Surprisingly, it was discovered here thatstandard shear forces experienced by the cells during washing reducedcell viability and resilience to cryopreservation.

In the new procedure, the contents of the cell factories harvested ateach passage are transferred into collection bags for cell washing. Anautomated cell washer (CytoMate® (Baxter, Deerfield, Ill.)) is usedduring processing to provide consistent, automated cell washing. Thechange in the washing process was to wash one collection bag at a time,and after completion of its wash cycle, transfer the contents of thewashed cells to a “Washed hMSC” collection (or holding) bag. Withoutbeing bound by theory, it is believed that this modification to theprocess reduced the cell wash exposure time per collection bag by onethird, thus protecting the cells from unnecessary handling and exposureto the shear forces of cell washing.

Example 29 Evaluation of Freezing Protocol

Based on low post-thaw cell viabilities observed in a development study,changes in the Cryomed® (Thermo Scientific, Rockford, Ill.) freezingprocess were made to ensure good cell viability and cell recoverypost-thaw. Surprisingly, standard procedures previously used forfreezing MSC preparations resulted in unacceptable cell viability.Changes in the Cryomed® (Thermo Scientific, Rockford, Ill.) freezingprocess included a modification to the freeze program so that it wouldclosely mimic what is experienced by cells which are simply placed in an−80° C. freezer inside an insulated container. These modificationsraised cell viability post-thaw by approximately 20%.

In manufacturing, a Cryomed (Thermo Scientific, Rockford, Ill.) freezeris used at the time of harvest to provide controlled-rate freezing ofthe cells to ≤−80° C. prior to the transfer to a LN₂ freezer (≤−135°C.). In development and research, small scale samples were initiallyfrozen directly in a −80° C. inside an insulated container, and thentransferred to a LN₂ freezer (≤135° C.). It was noted that these sampleshad consistently high viability. A study was conducted to map thetemperature profile under these conditions. This temperature profile wasthen programmed into the Cryomed (Thermo Scientific, Rockford, Ill.)freezer and tested. The effect was a more gradual temperature transitionto the final freezing temperature. This change was found to improve cellviability and was incorporated into the current Cryomed (ThermoScientific, Rockford, Ill.) freezing process used in manufacturing.

Example 30 Production of Clinical Scale MSC Preparations Through Passage2

Multiple clinical scale preparations were produced, each by the methodset forth in Example 1 through Example 15 to produce a collection ofpreparations. Each MSC preparation contained about 4.5×10⁹ to about6.3×10⁹ cells (about 30 to about 50 doses), for example about 5.5×10⁹cells derived from a single donor with an average of 5 populationdoublings compared to the P0 culture. After thawing, a sample from eachpreparation was tested for antigen profile, TNFRI profile, cellviability, and capacity for differentiation.

As shown in Table 6, each preparation in the collection had an antigenprofile of less than about 1.25% CD45+ cells, at least about 95% CD105+cells, and at least about 95% CD166+ cells. Also as shown in Table 6,each preparation in the collection had a TNFRI profile of at least about13 pg TNFRI per million cells. Each preparation had a cell viability ofat least about 70% (data not shown). Cells from each preparation weretested and determined to have a capacity for chondrogenesis,osteogenesis, and adipogenesis.

The antigen profile of MSCs was assessed by fluorescence-activated cellsorting (FACS) analysis. The assay used a single-antibody approach andgenerated data reflecting expression of the three markers individually(CD45−, CD105+, and CD166+). CD45 (LCA) is a marker for cells ofhematopoietic origin, while CD105 (endoglin) and CD166 (ALCAM) aremarkers for MSCs. The MSC samples were labeled with commercialfluorescent antibodies to the cell surface markers, then washed, fixedand subjected to flow cytometric analysis. An isotype antibody negativecontrol was also run at the same time. The MSC phenotype profile wasdetermined using CELLQuest software (Becton, Dickinson and Company, SanJose Calif.). The cells were first identified on the basis of lightscatter properties (forward scatter for size and side scatter forinternal granularity), while marker expression was assessed throughfluorescence from the bound antibodies. In the forward scatter versusside scatter dot blot for the unstained control, the main dot clusterwas positioned at the center of the side scatter axis by adjusting theside scatter voltage control. The acquisition gate, defined by the R1region, was subsequently adjusted to ensure that the main dot cluster iscompletely enclosed within the R1 region. The data acquisition processinvolved the accumulation of 10,000 events. When the analysis of theunstained control was complete, the isotype antibody negative controlwas analyzed using the acquisition parameters that were defined for theunstained control. The CD166, CD105, and CD45 test samples were analyzedusing the same approach. The fluorescence threshold for positive markerexpression was defined using the isotype antibody negative control,which was adjusted so that at least about 99.50% of the negative controlcells were identified as “negative”. This threshold was used to measurethe percentage of positive cells for each of the three cell surfacemarkers independently.

Cell viability was assessed by a flow cytometry of fluorescently stainedcells, based on differential staining of viable and non-viable cells ontheir respective permeabilities to DNA-binding dyes in a proprietaryreagent (Viacount™, Guava Technologies, Millipore, Billerica, Calif.).The stained sample was analyzed on a Guava PCA flow cytometer and theGuava CytoSoft software package (Millipore, Billerica, Calif.). TheGuava PCA uses a 532 nm diode laser to excite the dyes, whosefluorescence is detected by photomultipliers (PM: PM1 detects theviability dye emission at 580 nm; PM2 detects nuclear dye emission at675 nm), while a photodiode measures forward light scatter (FSC). Thefluorescence signal was resolved, allowing quantitation of the viableand non-viable cell populations from cellular debris in the testarticle.

To test for osteogenic differentiation, about 3×10 hMSCs were seededonto 35 mm dishes in medium (DMEM, 10% FBS). After 24 hours, the mediumwas replaced with osteogenic assay medium (DMEM, 10% FBS, 50 mMascorbate 2-phosphate, 10 mM p-glycerol phosphate, 0.1 mMdexamethasone), which was replaced every three to four days during the17-day period of the assay. At the end of culture period MSC osteogenicdifferentiation was assessed using von Kossa staining and by measurementof calcium accumulation in cells. Selected specimens were subsequentlystained for mineral by the von Kossa method. Cell layers were fixed with10% formalin for 1 hour, incubated with 2% (w/v) silver nitrate solutionfor 10 minutes in the dark, washed thoroughly with deionized water andthen exposed to bright light for 15 minutes. For the calcium assay, celllayers were rinsed twice with Phosphate Buffered Saline (PBS) andscraped off the dish in 0.5N Hydrochloric acid. Calcium was thenextracted from the cell layers by shaking for 4 hours at 4° C. followedby centrifugation at 1,000×g for 5 minutes. The calcium content of theresulting supernatant was determined according to the manufacturer'sinstructions for Sigma kit #587-A. Absorbance of samples was measured at575 nm using a Beckman spectrophotometer. Total calcium in samples wascalculated using standards assayed in parallel and expressed as μg perdish.

To test for chondrogenic differentiation, chondrogenic differentiationwas induced by gently pelleting about 2.5×10⁵ hMSCs in definedchondrogenic medium (high-glucose DMEM, ITS⁺ supplement(Becton-Dickinson), 0.1 mM dexamethasone, 10 ng/mL TGF-β3, 50 μg/mLascorbate 2-phosphate, 2 mM pyruvate+antibiotics) in a 15 mL conicaltube. The pellet culture was then incubated at 37° C. for 1 to 4 weekswith medium replacement every 2 to 3 days. At harvest, the pellets weregently fixed in 4% formaldehyde, embedded in paraffin, and then sectionscut and analyzed. Type II collagen was detected by immunohistochemicalmethods. Sections were also stained for proteoglycans using ToluidineBlue and Safronin O.

To test for adipogenic differentiation, adipogenic differentiation wasinduced by adding adipogenic induction medium (DMEM, 10% FBS, 1 mMdexamethasone, 0.5 mM methyl-isobutylxanthine, 0.2 mM indomethecin, 10μg/mL insulin and antibiotics) to hMSCs previously grown for severaldays after reaching confluency. After induction, the media was changedto adipogenic maintenance media (DMEM, 10% FBS, 10 μg/mL insulin andantibiotics) and the culture grown for a further 1 week. Lipid vacuolesin cells differentiated into adipocytes were detected by Oil Red O.

For the TNFRI assay, MSC samples were processed to lyse the cells andrelease TNFRI into solution. An ELISA method was used to quantify TNFRIusing a commercially available kit. This kit allowed for the measurementof both cell-associated TNFRI, and soluble TNFRI that has been shed fromcell surface. The assay employed a quantitative sandwich enzymeimmunoassay technique. Samples were added to a microplate that had beenprecoated with a monoclonal antibody specific for human TNFRI. TNFRIpresent in standards and samples was captured by the immobilizedanti-TNFRI antibody. After repeated washing to remove unbound extraneousmaterial, an enzyme-linked polyclonal antibody specific for TNFRI wasadded. Following incubation, a wash was done to remove unboundantibody-enzyme reagent, and then a substrate solution was added andcolor develops in proportion to the amount of TNFRI bound to themicroplate. The color development was stopped at a specified time pointand the intensity of the color is measured using a microplatespectrophotometer. Quantitation was achieved by comparing the signal ofunknowns to TNF RI standards assayed at the same time. Althoughquantification using this assay provides units of pg TNFRI/ml, theamount of TNFRI relative to the number of MSCs (‘potency’) can beobtained by calculating the concentration of MSCs before lysis.

TABLE 6 Clinical scale MSC preparations produced through passage 2Expected Test Sampling Test Method Results Lot 1 Lot 2 Lot 3 Lot 4 Lot 5Lot 6 Date of Manufacture 25 Aug. 26 Aug. 8 Sep. 29 Aug. 30 Aug. 9 Sep.2005 2005 2005 2005 2005 2005 Phenotype 2X DCB FACS   ≥95% 99.22% 99.28%99.42% 96.72% 98.05% 99.42% Formu- G-SOP- CD105 CD105 CD105 CD105 CD105CD105 CD105 lation Q0007   ≥95% 99.23% 99.35% 99.39% 98.37% 97.35%99.40% CD166 CD166 CD166 CD166 CD166 CD166 CD166 ≤1.25%  0.27%  0.00% 0.02%  0.10%  0.10%  0.01% CD45 CD45 CD45 CD45 CD45 CD45 CD45 TNF RIDCB Sandwich ≥108 pg/mL¹ 430 pg/mL 579 pg/mL 453 pg/mL 602 pg/mL 537pg/mL 372 pg/mL Formu- ELISA lation Cell Line DCB Isoenzyme human humanhuman human human human human Species Formu- electro- cell line cellline cell line cell line cell line cell line cell line Identity lationphoresis Karyology DCB cytogenic no no no no no no no Formu- evaluationchromosomal chromosomal chromosomal chromosomal chromosomal chromosomalchromosomal lation abnormalities abnormalities abnormalitiesabnormalities abnormalities abnormalities abnormalities Sterility 2X DCBUSP <71> Negative Negative Negative Negative Negative Negative NegativeFormu- Direct lation Method packaged DCB (post thaw) Mycoplasma pooled1993 FDA Negative Negative Negative Negative Negative Negative Negativesuspension PTC (Indirect and Direct method) Endotoxin 2X DCB USP <85>≤1.67 EU/mL pass pass pass pass pass pass Formu- (LAL; kinetic lationchromogenic) packaged DCB (post thaw) Ultra- DCB Thin- Negative NegativeNegative Negative Negative Negative Negatrve structural Formu- SectionEvaluation lation TEM of Cell Cultures for Viral Particles In Vitropooled MRC-5, Negative Negative Negative Negative Negative NegativeNegatrve Assay for suspension VERO, and Detection Hs68 cells ofAdventitious Virus Contaminants In Vitro pooled USFDA Negative NegativeNegatrve Negative Negative Negative Negative Assay for suspensionDetection Adventitious Virus Contaminates Detection 2X DCB DNA qPCRNegative Negative Negative Negative Negative Negative Negative of Formu-HTLV lation I & II Detection 2X DCB DNA qPCR Negative Negative NegatrveNegative Negative Negative Negatrve of Formu- Human lation HBV Detection2X DCB DNA qPCR Negative Negative Negative Negative Negative NegativeNegative of Formu- Human lation CMV Detection 2X DCB DNA qPCR NegativeNegative Negative Negative Negative Negative Negative of Formu- Humanlation EBV Detection 2X DCB DNA qPCR Negative Negative Negative NegativeNegative Negative Negative of Formu- HHV-6A lation and HHV-6B Detection2X DCB DNA qPCR Negative Negative Negative Negative Negative NegativeNegatrve of Formu- HIV-1 lation Detection 2X DCB DNA qPCR NegativeNegative Negative Negative Negative Negative Negatrve of Formu- HIV-2lation Detection 2X DCB qRT-PCR Negative Negative Negative NegativeNegative Negative Negative Parvovirus Formu- B-19 lation Detection 2XDCB DNA qPCR Negative Negative Negative Negative Negative NegativeNegative of Formu- HCV lation Detection DCB DNA qPCR Negative NegativeNegative Negative Negative Negative Negatrve of Formu- Human lationPapilloma Virus (HPV) Detection 2X DCB DNA qPCR Negative NegativeNegative Negative Negative Negative Negative of Formu- HHV-8 lation

Example 31 Production of Clinical Scale MSC Preparations Through Passage2

A collection of clinical scale preparations were produced and analyzed,as in Example 30. The results are depicted in Table 7, Table 8, andTable 9. As depicted, each preparation in the collection had an antigenprofile of less than 1.25% CD45+ cells, at least 95% CD105+ cells, andat least 95% CD166+ cells. Also as depicted, each preparation in thecollection had a TNFRI profile of at least 13 μg TNFRI per millioncells. Each preparation was tested and determined to have a cellviability of at least 70% (data not shown).

TABLE 7 Clinical Scale MSC Preparations Produced Through Passage 2 FACSFACS FACS IL- Mycoplasma CD166 CD105 CD45 Endotoxin³ TNF RI 2Rα P2 LotSterility¹ PTC (%) (%) (%) Sterility² (EU/mL) (pg/mL) (%) AppearanceNumber Negative Negative ≥95% ≥95% ≤1.25% Negative ≤1.67 ≥39 ≥30% Pass⁴ 7 Negative Negative  99% 99%   0.00% Negative <0.100/<0.100 80 55% Pass 8 Negative Negative  99% 99%   0.00% Negative <0.100 76 60% Pass   8⁵Positive N/A  99% 99%   0.40% Positive <1.00/<1.00 88 N/A Pass   9⁵Negative Negative  99% 99%   0.00% Positive <1.00/<1.00 87 N/A Pass 10Negative Negative  99% 99%   0.19% Negative <0.100/<0.100 100 57% Pass11 Negative Negative 100% 99%   0.00% Negative <0.100/<0.100 100 63%Pass 12 Negative Negative  99% 97%   0.00% Negative, <0.100/<0.100 12657% Pass Negative 13 Negative Negative  99% 98%   0.00% Negative,<0.100/<0.100 111 57% Pass Negative 14 Negative Negative  99% 98%  0.00% Negative, <0.100/<0.100 121 69% Pass Negative 15 NegativeNegative  99% 99% <0.50% Negative <0.100/<0.100 111 70% Pass 16 NegativeNegative  99% 99% <0.50% Negative <0.100 114 71% Pass  17⁵ NegativeNegative  99% 99% <0.50% Negative <1.00 112 76% Pass 18 NegativeNegative  99% 99% <0.50% Negative 0.177 128 83% Pass ¹Sterility testingis performed on the P2 2X intermediate Formulation sample. ²Sterilitytesting is performed on the final P2 packaged dose. Multiple testresults indicate two bags were pulled for testing: one from controlledrate freezer #1 and one from controlled rate freezer #2. Both resultsmust be negative for the lot to meet the sterility criterion. ³Endotoxintesting is performed on the final P2 packaged dose. Multiple testresults indicate two bags were pulled for testing-one from controlledrate freezer #1 and one from controlled rate freezer #2. Both resultsmust be ≤1.67 EU/mL for the P2 lot to meet the endotoxin criterion.⁴Pass = Opaque contents, off-white to pale amber in color. ⁵N/A = Notapplicable, no samples were available for testing due to microbialcontamination.

TABLE 8 Clinical Scale MSC Preparations Produced Through Passage 2 HHV-6Variant A and P2 Lot HCV CMV EBV HBV Variant B HHV-8 HIV-1 HIV-2 NumberNegative Negative Negative Negative Negative Negative Negative Negative 7 Negative Negative Negative Negative Negative Negative NegativeNegative  8 Negative Negative Negative Negative Negative NegativeNegative Negative   9¹ N/A N/A N/A N/A N/A N/A N/A N/A 10 NegativeNegative Negative Negative Negative Negative Negative Negative 11Negative Negative Negative Negative Negative Negative Negative Negative12 Negative Negative Negative Negative Negative Negative NegativeNegative 13 Negative Negative Negative Negative Negative NegativeNegative Negative 14 Negative Negative Negative Negative NegativeNegative Negative Negative 15 Negative Negative Negative NegativeNegative Neqative Negative Neqative 16 Negative Neqative NegativeNegative Negative Neqative Negative Neqative 17 Negative NegativeNegative Negative Negative Negative Negative Negative  18¹ NegativeNegative Negative Negative Negative Negative Negative Neqative 19Negative Negative Negative Negative Negative Negative Negative Negative¹N/A = Not applicable, no samples available for testing.

TABLE 9 Clinical Scale MSC Preparations Produced Through Passage 2In-Vivo Assay Thin for Section In-Vivo Adventitious Electron Assay ViralMicroscopy for Viral Contaminants for Cell Lines Contaminants (MRC-5,Detection Identity P2 Lot HTLV Parvovirus (mouse and VERO, Hs68 of ViralIsoenzyme Number I & II B-19 HPV 18 egg) Cells) ParticlesElectrophoresis Karyology Negative Negative Negative Negative NegatveNegative Human Cell No Lines Chromosomal Abnormalities  7 NegativeNegative Negative Negative Negative Negative Human Cell No LinesChromosomal Abnormalities  8 Negative Negative Negative NegativeNegative Negative Human Cell No Lines Chromosomal Abnormalities   9¹ N/AN/A N/A N/A N/A N/A N/A N/A 10 Negative Negative Negative NegativeNegative Negative Human Cell No Lines Chromosomal Abnormalities 11Negative Negative Negative Negative Negative Negative Human Cell NoLines Chromosomal Abnormalities 12 Negative Negative Negative NegativeNegative Negative Human Cell No Lines Chromosomal Abnormalities 13Negative Negative Negative Negative Negative Negative Human Cell NoLines Chromosomal Abnormalities 14 Negative Negative Negative NegativeNegative Negative Human Cell No Lines Chromosomal Abnormalities 15Negative Negative Negative Negative Negative Negative Human Cell NoLines Chromosomal Abnormalities 16 Negative Negative Negative NegativeNegative Negative Human Cell No Lines Chromosomal Abnormalities 17Negative Negative Negative Negative Negative Negative Human Cell NoLines Chromosomal Abnormalities  18¹ Negative Negative Negative NegativeNegative Negative Human Cell No Lines Chromosomal Abnormalities 19Negative Negative Negative Negative Negative Negative Human Cell NoLines Chromosomal Abnormalities ¹N/A = Not applicable, no test samplesavailable for testing.

Example 32 Production of Clinical Scale MSC Preparations Through Passage5

Multiple clinical scale preparations were produced, each by the methodset forth in Example 1 through Example 26, to produce a collection ofpreparations. Each preparation contained about 1×10¹² MSCs (up to about8,000 doses after a freeze-thaw cycle) derived from a single donor withan average of about 12.5 population doublings compared to the POculture, or 25 total doublings. After thawing, a sample from eachpreparation was tested for antigen profile, TNFRI profile, cellviability, and capacity for differentiation. With the exception of TNFRIprofile, each test was performed in the same manner described in Example30. For the TNFRI profile, a TNFRI assay was performed in the samemanner described in Example 30, and an assay of IL-2Rα expressioninhibition was also determined as described below.

Inhibition of IL-2Rα expression on T-cells was determined byco-culturing MSCs with CD3/CD28-activated hPBMCs for 3 days, the timecorresponding to the maximal level of IL-2Rα expression by the T-cellpopulation in hPBMCs. The ratio of hMSCs to hPBMCs is 1:5, which was theoptimal ratio identified for hMSC-mediated inhibition of lymphocyteproliferation tests. After incubation, cells were collected and lysed.The level of IL-2Rα in cell lysates was measured by ELISA. Results wereexpressed as % inhibition of IL-2Rα expression on CD3/CD28-stimulatedhPBMCs by hMSCs relative to the control CD3/CD28 hPBMC cultured withouthMSCs.

As shown in Table 10, each preparation in the collection had an antigenprofile of less than 1.25% CD45+ cells, at least 95% CD105+ cells, andat least 95% CD166+ cells. Also as shown in Table 10, each preparationin the collection had a TNFRI profile of 13 to 179 pg TNFRI per millioncells and inhibited IL-2Rα expression by at least 20% (at least 30%).Also as shown in Table 10, at least 70% (at least 80%) of the cells ineach preparation were viable. Cells from each preparation had a capacityfor chondrogenesis, osteogenesis, and adipogenesis (data not shown).

TABLE 10 Clinical scale MSC preparation produced through passage 5 20*21* 22* 23* 24* 25* DCB Derived From Lot 2 Lot 2 Lot 2 Lot 5 Lot 5 Lot 5Date of Manufacture 10 Mar. 17 Mar. 21 Mar. 20 Feb. 22 Feb. 23 Feb. 20062006 2006 2007 2007 2007 Manufacturing Manufacturer Test Step TestMethod OTI OTI OTI LONZA LONZA LONZA Sterility pooled Negative NegativeNegative Negative Negative Negative Negative suspension packaged PDNegative Negative Negative Negative Negative Negative (post-thaw)Mycoplasma pooled Negative Negative Negative Negative Negative NegativeNegative suspension Endotoxin pooed ≤1.67 EU/mL <0.050 0.050 <0.050<0.050 <0.050 O.050 suspension packaged PD <0.25 <0.25 <0.025 <0.25<0.050 <0.050 (post-thaw) Viability packaged PD ≥70% viable cells 88 8486 92 94.8 86 (post-thaw) Phenotype 2X PD ≥95% CD105 ≥98.91% ≥98.17%≥98.52% ≥99.53% ≥99.52% ≥99.602% (Identity/ Formulation ≥95% CD166 CD105CD105 CD105 CD105 CD105 CD105 Purity) ≤1.25% CD45**  ≥98.49% ≥95.59%≥97.84% ≥98.95% ≥99.51% ≥99.47% CD166 CD166 CD166 CD166 CD166 CD166 ≤0.00%  ≤0.04%  ≤0.45%  ≤0.03%  ≤0.00%  ≤0.00% CD45 CD45 CD45 CD45 CD45CD45 TNFRI PD Min: 272 pg/mL** 961 900 963 995 1269 1162 (potency)Formulation Max: 1471 pg/mL IL-2Ra PD ≤30% inhibition of N/A N/A N/A 3458 62 (potency) Formulation II-2Ra expression Residual 2X PD ≤10.0ug/mL** 3.0 1.08 3.05 3.11 5.60 2.59 BSA Formulation Residual 2X PD≤30.0 ug/mL** 21.21 21.3 <14.4 <14.4 14.4 <14.4 Trypsin FormulationAppearance** Packaged PD Opaque, off-white N/A N/A N/A Pass Pass Pass(cells in bag) (post-thaw) to pale amber, no fine particulates, no cellclumping***

Example 33 Production of Clinical Scale MSC Preparations Through Passage5

A collection of clinical scale preparations were produced and analyzed,as in Example 32.

Table 11 depicts the results of the preparation screen for theproperties listed in Table 12 and the population density in the CFs. Asdepicted in Table 11, all preparations successfully analyzed passed theexamplary release criteria.

As depicted in Table 12, each preparation in the collection had anantigen profile of less than 1.25% CD45+ cells, at least 95% CD105+cells, and at least 95% CD166+ cells.

Also as depicted in Table 12, each preparation in the collection had aTNFRI profile of at least 13 pg TNFRI per million cells.

Also as depicted Table 12, each preparation in the collection had aTNFRI profile of at least 13 pg TNFRI per million cells and inhibitedIL-2Rα expression by at least about 20% (at least about 30%). Eachpreparation had a cell viability of at least about 70% (at least about80%).

In order to determine if preparations could consistently be produced,the variance and standard deviation in the antigen profile, TNFRIprofile, and cryopreservation profile were calculated as measures ofdeviation within the collection set forth in Table 12. The results areshown at the end of Table 12. As seen in Table 12, there is minimaldeviation in the profiles (note that the cutoff value (preceded by a “<”symbol) was used for deviation calculation in certain instances whereactual values were not determined).

TABLE 11 Clinical Scale MSC Preparations Through Passage 5 Released ifAverage Total lot meets Viable Cells/Cell P5 Lot P2 Lot Used toexamplary Factory at Number Manufacture DP criteria Harvest 26 7Released 174 × 10⁶ 27 14 Released 168 × 10⁶ 28 14 Released 191 × 10⁶ 2913 Released 122 × 10⁶ 30 13 Released 156 × 10⁶ 31 13 Released 181 × 10⁶32 13 N/A N/A 33 13 N/A N/A 34 14 Released 179 × 10⁶ 35 14 Released 121× 10⁶ 36 14 Released 151 × 10⁶ 37 14 Released 121 × 10⁶ 38 14 Released164 × 10⁶ 39 14 Released 164 × 10⁶ 40 14 Released 172 × 10⁶ 41 14Released 201 × 10⁶ 42 14 Released 160 × 10⁶ 43 14 Released 142 × 10⁶ 4414 Released 210 × 10⁶ 45 14 Released 237 × 10⁶ 46 14 Released 195 × 10⁶47 14 Released 173 × 10⁶ 48 14 Released 190 × 10⁶ 49 14 Released 168 ×10⁶ 50 14 Released 173 × 10⁶ 51 14 Released 162 × 10⁶ 52 8 Released 147× 10⁶ 53 14 Released 169 × 10⁶ 54 14 Released 147 × 10⁶ 55 14 Released166 × 10⁶ P5 lots 32 and 33 were rejected prior to harvest due to an inprocess error. No QC samples were generated.

TABLE 12 Clinical Scale MSC Preparations Produced through Passage 5 TNFRI Myco- Endo- Post Residual Residual (pg/mL) P5 Steril- plasma FACSFACS FACS Steril- toxin Thaw BSA Trypsin Min: IL- Ap- Lot ity¹ PTC CD166CD105 CD45 ity² (EU/mL) Viability (ug/mL) (ug/mL) 108 2Rα pear- Num-Nega- Nega- (%) (%) (%) Nega- ≤1.67 (%) ≤10 ≤30 Max: (%) ance ber tivetive ≥95% ≥95% ≤75% tive EU/mL ≥70% ug/mL ug/mL 368 ≥30% Pass³ 26 Nega-Nega- 98 98 <0.50 Nega- 0.421 96 2 <28.8 179 75 Pass tive tive tive 27Nega- Nega- 99 99 <0.50 Nega- <0.250 92 2 <14.4 217 76 Pass tive tivetive 28 Nega- Nega- 99 99 0.71 Nega- <0.250 92 1 <14.4 252 79 Pass tivetive tive 29 Nega- Nega- 98 98 <0.50 Nega- <0.250 92 1 <28.8 297 77 Passtive tive tive 30 Nega- Nega- 99 99 <0.50 Nega- <0.250 93 2 <14.4 232 76Pass tive tive tive 31 Nega- Nega- 98 99 0.67 Nega- <0.250 93 2 <14.4252 76 Pass tive tive tive 34 Nega- Nega- 98 96 0.55 Nega- <0.250 93 1<14.4 247 80 Pass tive tive tive 35 Nega- Nega- 98 98 <0.50 Nega- <0.25091 1 <14.4 340 75 Pass tive tive tive 36 Nega- Nega- 95 96 <0.50 Nega-<0.250 90 1 <14.4 314 79 Pass tive tive tive 37 Nega- Nega- 98 98 <0.50Nega- <0.250 86 2 <14.4 270 79 Pass tive tive tive 38 Nega- Nega- 97 97<0.50 Nega- <0.250 83 2 <14.4 318 78 Pass tive tive tive 39 Nega- Nega-97 98 <0.50 Nega- <0.250 87 1 <14.4 286 79 Pass tive tive tive 40 Nega-Nega- 99 99 <0.50 Nega- <0.250 86 2 <14.4 299 78 Pass tive tive tive 41Nega- Nega- 98 98 <0.50 Nega- <0.250 90 1 <14.4 240 82 Pass tive tivetive 42 Nega- Nega- 98 98 <0.50 Nega- <0.250 90 2 <14.4 286 74 Pass tivetive tive 43 Nega- Nega- 97 98 <0.50 Nega- <0.250 88 2 <14.4 321 67 Passtive tive tive 44 Nega- Nega- 97 98 <0.50 Nega- <0.250 87 2 <14.4 340 74Pass tive tive tive 45 Nega- Nega- 97 98 <0.50 Nega- <0.250 89 2 <14.4336 77 Pass tive tive tive 46 Nega- Nega- 98 98 <0.50 Nega- 0.286 91 2<14.4 340 78 Pass tive tive tive 47 Nega- Nega- 98 99 <0.50 Nega- 0.30280 2 <14.4 299 75 Pass tive tive tive 48 Nega- Nega- 98 98 <0.50 Nega-<1.00 85 2 <28.8 314 85 Pass tive tive tive 49 Nega- Nega- 99 99 <0.50Nega- 0.550 93 2 <14.4 335 76 Pass tive tive tive 50 Nega- Nega- 99 99<0.50 Nega- 0.730 90 3 <28.8 306 85 Pass tive tive tive 51 Nega- Nega-99 99 <0.50 Nega- <0.250 92 3 <14.4 304 79 Pass tive tive tive 52 N/ANega- 99 99 <0.50 Nega- <0.250 94 2 <14.4 300 67 Pass tive tive 53 Nega-Nega- 97 98 <0.50 Nega- <0.250 94 2 <14.4 323 78 Pass tive tive tive 54Nega- Nega- 99 99 <0.50 Nega- 0.370 91 3 <14.4 301 81 Pass tive tivetive 55 Nega- Nega- 98 99 0.64 Nega- <0.250 93 4 <14.4 328 71 Pass tivetive tive 56 Nega- Nega- 98 99 <0.50 Nega- <0.250 92 3 <14.4 277 68 Passtive tive tive 57 Nega- Nega- 98 99 <0.50 Nega- 0.365 93 4 <14.4 313 71Pass tive tive tive 58 Nega- Nega- 99 100 <0.50 Nega- 0.536 91 3 <14.4330 69 Pass tive tive tive 59 Nega- Nega- 95 96 <0.50 Nega- <0.250 88 2<14.4 334 67 Pass tive tive tive 60 Nega- Nega- 99 100 <0.50 Nega-<0.250 94 3 <14.4 273 66 Pass tive tive tive 61 Nega- Nega- 99 100 <0.50Nega- <0.250 86 2 <28.8 333 76 Pass tive tive tive 62 Nega- Nega- 99 99<0.50 Nega- <0.250 92 3 <14.4 359 75 Pass tive tive tive Vari- 1.0873951.011765 0.002524 12.16471 0.633613 1648.706 23.99664 ance Stand-1.042782 1.005865 0.05024 3.487794 0.795998 40.60426 4.898636 ard Dev

Example 34 Analysis of Profile Deviation

In order to determine if preparations could consistently be produced,the variance and standard deviation in the antigen profile, TNFRIprofile, and cryopreservation profile were calculated as measures ofdeviation within the collection set forth in Table 10. The results areshown in Table 13. As seen in Table 13, there is minimal deviation inthe profiles.

TABLE 13 Consistent Production of MSC Preparations Cryo- preser- TNFRIProfile vation Antigen Profile % Profile Prep- % % % IL-2Ra % arationCD105+ CD166+ CD45+ TNFRI inhibition Viability 20 98.91 98.49 0.00 961NA 88 21 98.17 95.59 0.04 900 NA 84 22 98.52 97.84 0.45 963 NA 86 2399.53 98.95 0.03 995 34 92 24 99.52 99.51 0.00 1,269 58 94.8 25 99.6099.47 0.00 1,192 62 86 Vari- 0.31 1.81 0.027 18,200 152.9 14.2 anceStand- 0.552347 1.34455 0.163 134.8 12.36 3.76 ard Dev

Example 35 Consistent Proliferation

An analysis of cellular proliferation was used to assess processconsistency, although cells from different donors will have differentproliferation potential. The evaluation of the total cells harvested foreach MSC preparation also allows for the identification of any out oftrend values. One measure of process consistency is the number of cellsper CF, since it reflects the uniformity of the process performance.However, due the inherent biological variability of the preparation of acellular product, the process will always be subject to a certain levelof variability.

The number of cells recovered per CF was evaluated at two passagesduring the manufacture of MSC preparations. The number of cells per CFafter the third passage (see Example 17) and the fifth passage (seeExample 23) for several MSC preparations is presented with descriptivestatistics in Table 14. At P3, the average number of cells per CF wasabout 190 million, and the standard deviation was 67. At P5, the averagenumber of cells per CF was about 166 million, and the standard deviationwas 35.

TABLE 14 Cellular Proliferation DCB Passage 3 (P3) Cells/ Harvest (P5)Cells/ PD Lot # Lot # CF (in millions) CF (in millions) 63 63 238 154 6463 116 141 65 63 116 126 66 63 315 148 67 63 157 117 68 63 154 141 69 2122 188 70 1 71 162 71 1 143 140 72 3 118 124 73 3 134 102 74 2 163 18975 2 229 238 76 2 229 197 77 2 246 208 78 2 246 174 79 2 202 218 20 2202 153 80 2 194 180 21 2 194 172 81 2 295 185 22 2 295 186 Mean 190 166Std. Dev. 67 35

Example 36 Evaluation of Cryoprotectant Fill Time

Product formulation and bag fill time ranges for an examplary ex vivocultured mesenchymal stem cells manufacturing process was performed toevaluate the effects of the cryoprotectant solution on cells todetermine an examplary time range for formulation and fill prior tocryopreservation. Four time points were evaluated: 60, 90 (control), 150and 240 minutes. At one day and thirty days post-cryopreservation, hMSCswere thawed and evaluated for viability (Table 15), phenotype (Table 16)and expression of TNFRI (potency) (Table 17) against this examplaryacceptance criteria. Results of the study showed that cell quality isnot affected, for example, when product formulation and fill occurswithin 150 or 240 minutes prior to cryopreservation.

TABLE 15 Effect of Different Formulation/Fill Times on Post-Thaw CellViability Example Acceptance Post-Thaw Criteria for Post-Thaw Cell CellViability Time for Formulation/Fill Viability (%) (%) One Day StorageAfter Cryopreservation  60 minutes ≥70 87.3 90 minutes (control) ≥7087.6 150 minutes ≥70 85.3 240 minutes ≥70 90.5 30 Days Storage AfterCryopreservation  60 minutes ≥70 87.4 90 minutes (control) ≥70 80.3 150minutes ≥70 92.1 240 minutes ≥70 88.8

TABLE 16 Effect of Different Formulation/Fill Times on Cell PhenotypePositively stained cells (%) Example Example Example CD166 CD105 CD45(%) (%) (%) Time for Accept- Accept- Accept- Formu- ance CD166 anceCD105 ance CD45 lation/Fill Criteria (%) Criteria (%) Criteria (%) OneDay Storage After Cryopreservation  60 minutes ≥95 98.28 ≥95 99.07 ≤0.750.37  90 minutes ≥95 99.19 ≥95 99.47 ≤0.75 1.57× (control) 150 minutes≥95 98.58 ≥95 99.08 ≤0.75 0.11 240 minutes ≥95 97.97 ≥95 98.99 ≤0.750.26 30 Days Storage After Cryopreservation  60 minutes ≥95 96.07 ≥9598.08 ≤0.75 0  90 minutes ≥95 96.75 ≥95 98.81 ≤0.75 0.21 (control) 150minutes ≥95 96.12 ≥95 98.56 ≤0.75 0 240 minutes ≥95 95.21 ≥95 98.62≤0.75 0 ×Investigation showed that the out of specification result wasnot a result of the test condition.

TABLE 17 Effect of Different Formulation/Fill Times on TNF RI ExpressionExample Acceptance TNF RI Criteria Expression Time for Formulation/FillTNFRI (pg/mL) (pg/mL) One Day Storage After Cryopreservation  60 minutes108 ≤ TNFRI ≤ 368 188 90 minutes (control) 280 150 minutes 258 240minutes 267 30 Days Storage After Cryopreservation  60 minutes 108 ≤TNFRI ≤ 368 318 90 minutes (control) 298 150 minutes 312 240 minutes 368

Example 37 Karyotyping

The karyotype of an hMSC sample is determined through examination ofcolcemid-arrested metaphase spreads. The hMSCs are cultured briefly,arrested with colcemid and processed. The cells are enlarged using ahypotonic HEPES buffered EGTA solution, fixed, and then the cells areenlarged using a hypotonic HEPES buffered EGTA solution, fixed, and thenplaced on microscope slides. The slides are aged overnight, treated withtrypsin, and stained with Giemsa to produce G-banding. Typical G-bandingshould yield about 350-450 positive G bands. Standard evaluationincludes enumeration of numerical and structural abnormalities(‘chromosomal abnormalities’), such as translocations, breaks, rings,markers and double-minutes, with at least 20 cells evaluated. Inversion,deletion, or translocation breakpoints are identified as per the ISCN1995 International System for Human Cytogenetic Nomenclature, whenpossible. At least two karyotypes per sample are prepared. Results ofkaryotyping show that examplary preparations of the present inventionhave no chromosomal abnormalities (Table 4, Table 6, and Table 9).

Example 38 Pathogen Contaminants

Testing for Adventitious Agents.

The preparations of the present invention can be made without beingcompromised by adventitious agents (pathogens). A useful preparation isoptionally screened for the following pathogens.

Sterility.

The sterility of the test article is determined by the DirectInoculation Method (Direct Method) in accordance with <USP 71> SterilityTest. The test conforms to the standard 14-day incubation period underaerobic and anaerobic conditions. Fluid Thioglycollate Medium is usedfor the culture of anaerobic bacteria and Soybean-Casein Digest (SCD)Medium is used for both fungi and aerobic bacteria. A test Negativeresult is no growth of microorganisms after 14 days incubation.Validation of the test method includes the following strains of testmicroorganisms: FTM (anaerobic)-[B. subtillis (ATCC 6633, K. rhizophilia(ATCC 9341), and C. sporogenes (ATCC 11437)] and SCD (aeorobic andfungi)-[B. subtillis (ATCC 6633); C. albicans (ATCC 10231), and A. niger(ATCC 16404)].

Mycoplasma.

Both indirect and direct methods are used.

i. The indirect method allows visualization of mycoplasma in aco-cultured indicator cell line (Vero cells). The test article hMSCs areco-cultured with Vero cells on coverslips. Positive controls for thetest are Vero cells inoculated with the Mycoplasma species M. hyorhinisand M. orale. The negative control is Vero cells inoculated with sterilebroth. Mycoplasmas are identified by epiflorescent detection of aDNA-binding fluorochrome stain (Hoechst stain). In negative controls,only the Vero cell nucleus is stained, whereas in positive controls,both nuclear and extra-nuclear fluorescence is detected. Mycoplasmaexhibit a staining pattern that differs from the host Vero cells, andconsists of extra-nuclear staining of small round bodies ofapproximately 0.3 μm in diameter.

ii. The direct method allows detection of mycoplasma growth underaerobic and anaerobic conditions (broth culture flasks and agar plates,respectively). The agar and broth media supply nutrients and a source ofcarbon and energy necessary for mycoplasma growth. Positive controlsconsist of cultures inoculated with nonfermentative and fermentativemycoplasma species (M. orale and M. pneumoniae, respectively), whilesterile broth serves as a negative control. Indicators of mycoplasmagrowth include change in color or turbidity of broth cultures, andgrowth of colonies with “fried egg” morphology on agar plates.

Negative results for mycoplasma contamination of the hMSC test sampleare defined as results for both indirect and direct methods thatresemble the respective negative controls. This test is governed by anOTI-approved protocol executed by an external testing laboratory (AppTecprotocol 30055).

Endotoxin.

Endotoxin levels in the test article are determined by the LimulusAmebocyte Lysate (LAL) method in accordance with <USP 85>. This test isa quantitative, chromogenic kinetic assay for the detection ofgram-negative bacterial endotoxin. The concentration of endotoxin in thetest article is calculated from a standard curve using a series ofendotoxin standards. The assay is validated to ensure the test articledoes not inhibit or enhance detection of endotoxin. A positive productcontrol (PPC) is included to ensure the sample dilution is valid.

Ultrastructural Evaluation of Cell Cultures for Viral Particles.

The ultrastructural morphology of an hMSC sample will be evaluated bythin-section transmission electron microscopy (TEM) to determine thefollowing:

Presence of a variety of viral types including retroviruses,herpesviruses, adenoviruses, picornaviruses, parvoviruses,orthomyxoviruses, paramyxoviruses, and reoviruses.

Presence of other microbial agents such as yeast, fungi or bacteria.

Incidence of specific retroviral morphologies (A-, B- C-, D- and R-typeparticles).

The test article is cultured briefly and the harvested. The cells areharvested, fixed, embedded, and sectioned for analysis by TEM. Twohundred cells are evaluated for any particle with virus-like morphology.A tabulation of retrovirus-like particles is undertaken. A blank watersample serves as the negative control.

In Vivo Test for Detection of Adventitious Viruses (Viral Contaminants)

The in-vivo viral test assay is designed to detect murine and non-murineviruses that would not be detected by the in vitro viral test. The assaysensitivity is increased by subpassage of materials from mice and eggsinto new test systems. The test article is prepared as a clarifiedlysate supernatant and inoculated into the following:

Adult mice (intraperitoneal and intracerebral administration). Adultmice are susceptible to various viral agents (includingcoxsackieviruses, flaviviruses). Daily health observations of the adultmice are performed and they are sacrificed 21 days after inoculation, orpossibly sooner if group mortality exceeds 20%.

Newborn suckling mice (intraperitoneal and intracerebraladministration). Suckling mice are susceptible to various agents,including togaviruses, bunyaviruses, flaviviruses, picornaviruses, andherpes viruses. The newborn suckling mice are observed for 14 days postadministration for ill effects, or possibly sooner if group mortalityexceeds 20%. At that time the mice are sacrificed, their tissues pooled,and homogenized for subpassage into another set of newborn suckling micethrough the same routes of administration. This group is monitored for14 days. Animals found dead within 24 hours of injection have theirtissues homogenized and sub-passaged into a fresh group of animals.

Embryonated hen eggs (allantois and yolk sac administration) is used totest for viruses. Inoculation via the allantois favors propagation oforthomyxoviruses, and paramyxoviruses. Inoculation via the yolk sacfavors replication of herpes viruses, rickettsiae, mycoplasma, andbacteria.

Embryonated hen's eggs inoculated via the allantoic cavity (firstpassage) are incubated for three days and the embryos are observed forviability. Allantoic fluids from this first passage are harvested(including fluids from dead eggs) for a hemagglutination assay (toquantify viral particles) or for sub-passage into a fresh set of eggs.The second passage eggs follow the same time course, at the end of whichthe allantoic fluids are harvested for a hemagglutination assay and anexamination of the embryos.

Eggs inoculated via the yolk sac (first passage) are incubated for ninedays and are observed for viability. After the 9-day incubation, allembryos are harvested and examined. All first passage yolk sacs areharvested, washed, and pooled; second passage eggs are inoculated with apreparation of this material. Second passage eggs, along with thenegative control, are incubated for 9 days and are candled each workingday for viability. After the 9 day incubation all embryos are harvestedand examined.

The hemagglutination assay uses chicken, guinea pig, and human type-0erythrocytes. Replicate plates are observed after incubation forhemagglutination.

A negative control is prepared for each set in the study group with aninoculation of Eagle's Minimal Essential Medium and processed andanalyzed in the same manner as the study group. A test article isconsidered negative for adventitious agents if: 1) at least 80% of adultand suckling mice (first and second passage) survive the test period ingood health; 2) at least 80% of inoculated hen embryos survive the testperiod and are normal in appearance; and 3) the fluids collected frominoculated embryos do not produce hemagglutination.

In Vitro Test for Detection of Adventitious Viruses.

The in vitro viral test is designed to detect various viruses, includingpicornaviruses, orthomyxoviruses, paramyxoviruses, herpesviruses,adenoviruses, and reoviruses. Test article is incubated with threeindicator cell lines (MRC-5, a human embryonic cell line; VERO, a simiankidney cell line; Hs68, a human foreskin cell line). The inoculatedcultures are examined for at least 28 days and compared to positivecontrols for the development of cell morphology characteristic of viralcontamination. Picornaviruses, herpesviruses, adenoviruses, andreoviruses can be detected by changes in morphology directly. However,orthomyxo- and paramyxo-viruses may replicate in these cell lines withlittle or no cytopathic manifestation. The presence of these viruses isdetected by the ability to adsorb erythrocytes to the surface ofinfected cells (hemadsorption assay) which is performed at theconclusion of the observation period.

HTLV I & II.

Testing for human T-cell lymphotropic viruses I and II (HTLV-I andHTLV-II) is designed to detect the presence of HTLV-1 and HTLV-2proviral DNA tax gene sequences in test article. The assay limit ofdetection is 10-100 infected cell equivalents of HTLV I or HTLV II DNA.The test method is based upon PCR amplification of a conserved region ofthe viral genome containing the tax/rex locus (the tax and rex lociencode proteins essential for viral replication). The specific ampliconis detected through use of a FITC-labeled donor probe and anLC640-labeled acceptor probe to provide a quantitative, real-time,sequence-specific analysis using a Roche LightCycler™. The fluorescenceis detected and correlates to viral DNA copy number. A negative resultis defined as a fluorescence signal below the limit of detection (lowestcopy number detected above background).

HBV.

The human hepatitis B virus (HBV) assay is a limit test designed todetect the presence of HBV DNA sequences, down to 10-100 copies of HBVviral DNA in the test article. The test method is based upon PCRamplification of the S region of the HBV genome, yielding a 194 bpamplification product. The specific amplicon is detected through use ofa FITC-labeled donor probe and an LC640-labeled acceptor probe toprovide a quantitative, real-time, sequence-specific analysis using aRoche LightCycler™. The fluorescence is detected and correlates to viralDNA copy number. A negative result is defined as a fluorescence signalbelow the limit of detection (lowest copy number detected abovebackground).

CMV.

The human cytomegalovirus (CMV) assay is a limit test designed to detectthe presence of CMV DNA sequences (as few as 10-100 DNA copies) in thetest article. The test method is based upon PCR amplification of aregion of the viral genome containing a highly conserved Hind III-Xfragment, yielding a 311 bp amplification product. The specific ampliconis detected through use of a FITC-labeled donor probe and anLC640-labeled acceptor probe to provide a quantitative, real-time,sequence-specific analysis using a Roche LightCycler™. The fluorescenceis detected and correlates to viral DNA copy number. A negative resultis defined as a fluorescence signal below the limit of detection (lowestcopy number detected above background).

EBV.

The human Epstein-Barr virus (EBV) assay is a limit test designed todetect the presence of EBV gene sequences (as few as 10-100 EBV DNAcopies) in the test article. The test method is based upon PCRamplification of a region of the EBV major capsid protein (MCP) gene,yielding a 212 bp amplification product. The specific amplicon isdetected through use of a FITC-labeled donor probe and an LC640-labeledacceptor probe to provide a quantitative, real-time, sequence-specificanalysis using a Roche LightCycler™. The fluorescence is detected andcorrelates to viral DNA copy number. A negative result is defined as afluorescence signal below the limit of detection (lowest copy numberdetected above background).

HHV-6 Variant a (HHV-6A) and Variant B (HHV-6B).

The assay for human herpesvirus 6, variant A (HHV-6A) and variant B(HHV-6B) is a limit test designed to detect the presence of HHV-6A andHHV-6B DNA sequences in the test article (as few as 10-100 copies ofHHV-6A and 6B DNA). The test method is based upon PCR amplification of aregion of the viral genome encompassing the U65-U66 genes, yielding a196 bp amplification product. The primers were derived from HHV-6A butalso amplify an identical sequence in HHV-6B. The specific amplicon isdetected through use of a FITC-labeled donor probe and an LC640-labeledacceptor probe to provide a quantitative, real-time, sequence-specificanalysis using a Roche LightCycler™. The fluorescence is detected andcorrelates to viral DNA copy number. A negative result is defined as afluorescence signal below the limit of detection (lowest copy numberdetected above background).

HIV-1.

The human immunodeficiency virus type 1 (HIV-1) assay is a limit testdesigned to detect the presence of HIV-1 gag region DNA sequences in thetest article (as few as 10-100 HIV-1 DNA copies). The test method isbased upon PCR amplification of a region of the viral genomeencompassing the gag locus, yielding a 140 bp amplification product. Thespecific amplicon is detected through use of a FITC-labeled donor probeand an LC640-labeled acceptor probe to provide a quantitative,real-time, sequence-specific analysis using a Roche LightCycler™. Thefluorescence is detected and correlates to viral DNA copy number. Anegative result is defined as a fluorescence signal below the limit ofdetection (lowest copy number detected above background).

HIV-2.

The human immunodeficiency virus type 2 (HIV-2) assay is a limit testdesigned to detect the presence of HIV-2 gag region DNA sequences in thetest article (as few as 50-500 infected cell equivalents of HIV-2 viralDNA). The test method is based upon PCR amplification of a region of theviral genome, encompassing the gag locus, yielding a 196 bpamplification product. The specific amplicon is detected through use ofa FITC-labeled donor probe and an LC640-labeled acceptor probe toprovide a quantitative, real-time, sequence-specific analysis using aRoche LightCycler™. The fluorescence is detected and correlates to thenumber of infected cell equivalents. A negative result is defined as afluorescence signal below the limit of detection. This test is governedby an OTI-approved protocol executed by an external testing laboratory.

Parvovirus B-19.

The parvovirus B-19 assay is a limit test designed to detect thepresence of parvovirus B-19 VP1 region DNA sequences in the test article(as few as 10-100 B-19 viral DNA copies). The test method is based uponPCR amplification of a region of the viral genome, within the VP1 gene,yielding a 208 bp amplification product. The specific amplicon isdetected through use of a FITC-labeled donor probe and an LC640-labeledacceptor probe to provide a quantitative, real-time, sequence-specificanalysis using a Roche LightCycler™. The fluorescence is detected andcorrelates to viral DNA copy number. A negative result is defined as afluorescence signal below the limit of detection (lowest copy numberdetected above background).

HCV.

The human hepatitis C virus (HCV) assay is a limit test designed todetect the presence of HCV RNA sequences in the test article (as few as1-10 HCV virus genomes). The test method is based upon reversetranscriptase-mediated synthesis of DNA complementary to viral RNApresent in the test article, followed by PCR amplification of the highlyconserved 5′ untranslated region of the HCV genome, yielding a 297 bpamplification product. The specific amplicon is detected through use ofa FITC-labeled donor probe and an LC640-labeled acceptor probe toprovide a quantitative, real-time, sequence-specific analysis using aRoche LightCycler™. The fluorescence is detected and correlates to viralcopy number. A negative result is defined as a fluorescence signal belowthe limit of detection (lowest copy number detected above background).

HPV Type 18.

This is a new assay implemented post Q4 2006 to replace the HPV assay.The human papillomavirus type 18 (HPV 18) assay is a limit test designedto detect viral DNA in the test article (as few as 1-10 copies/infectedcell equivalent (ICE)/plaque-forming unit (Pfu). The test method isbased upon FOR amplification of a region of the viral genome, within theE6/E7 region, yielding a 208 bp amplification product. The specificamplicon is detected using a quantitative, real-time, sequence-specificanalysis using a Roche LightCycler™. The presence of amplified HPVtarget DNA is confirmed by DNA probe specific hybridisation with afluorescence-labeled HPV oligonucleotide probe that identifies aninternal sequence of 208 bp Amplicon. The fluorescence is detected andcorrelates to viral particle number. A negative result is defined as afluorescence signal below the limit of detection (lowest viral particlenumber detected above background).

HHV-8.

The human herpesvirus 8 (HHV-8) assay is a limit test designed to detectthe presence of HHV-8 IE1A gene DNA sequences (10-100 infected cellequivalents of HHV-8 DNA) in test article. The test method is based uponPCR amplification of a region of the viral genome, encompassing the IE1Agene, yielding a 264 bp amplification product. Amplified reactionproducts are then fractionated on an agarose gel, blotted by theSouthern method onto a DNA binding membrane, and probed with aradiolabeled oligonucleotide homologous to a DNA sequence embeddedwithin the 264 bp-amplified product. Amplicon-specific radioactivity ismeasured by autoradiography.

Example 39 Aseptic Processing

Certain steps occurred throughout the manufacturing process to controlthe environment. To reduce potential contamination, the Terumo® SterileTubing Welder and the Sebra® MINI™ Hand Held Tube Sealer were utilizedto permit closed processing. Steps that used the welder and sealerincluded: transfer of BMA and fluids, sampling, seeding of CFs, feeds,passages, harvest, formulation, fill and cryopreservation. All welds andseals were visually inspected for integrity. If the integrity of a sealor weld has been breached (exposed to air), then the affected portion ofthe material will be discarded.

Example 40 Control of Tube Welding for Aseptic Processing

In all processing steps, tube to tube welding (cell factories (CFs),solution bags and cryopreservative (e.g., Cryocyte™) freezingcontainers) was surprisingly useful to implement controls to reducepotential contamination. All welding of tubing was performed using theTerumo® Sterile Tubing Welder. This welder has been designed for use inthe storage and handling of blood products. The system automaticallyconnects in a sterile manner two sections of tubing. The welds werevisually inspected to ensure integrity by rotating the tubing 360° todetermine if the outer diameters of the two tubes were aligned at theconnection (weld). If the welds were determined to be breached (exposedto air), then the affected portion of material was discarded. Ifproperly aligned, each weld was opened by pinching or rolling the tubinguntil the fluid pathway opens. Process steps that are surprisinglyenhanced by welding include, for example, transfer of BMA and fluids,sampling, seeding of CFs, feeds, passages, harvest, formulation andfill.

Example 41 Control of Tube Seals for Aseptic Processing

Processing steps that require tubing to be sealed or severed utilizedthe Sebra® MINI™ Hand Held Tube Sealer. The tube sealer is an instrumentfor making seals on tubing made of Radio Frequency (RF)-reactivethermoplastic materials used in blood banks, blood processing facilitiesand transfusion centers. The sealer is an instrument that generates acontrolled amount of RF energy at the sealing head while mechanicallycompressing the tubing across its diameter during the dielectric sealingand forming process. When the energy is removed and the tubing isallowed to cool under compression, a permanent seal is produced. Duringprocessing, the seals were inspected visually for integrity. If a sealwas determined to be breached (exposed to air), that portion of materialwas discarded. Process steps that are surprisingly enhanced by sealsinclude transfer of BMA and fluids, sampling, seeding of CFs, feeds,passages, harvest, formulation, fill and cryopreservation.

Example 42 Therapeutic Efficacy of Clinical Scale Preparations

A clinical scale preparation was prepared as detailed in Example 1through Example 26. The preparation was partitioned into therapeuticdoses and administered to pediatric aGVHD patients.

Eight biweekly therapeutic doses of 2×10⁶ cells/kg were administered byinfusion to 59 children with steroid (and other therapeuticagent)-refractory Grade B-D aGVHD. An additional 4 weekly infusions wereadministered after day 28 in patients who had a response.

At baseline, the distribution of aGVHD grades B:C:D was 6 (10%): 20(33%): 33 (57%). The median duration of aGVHD before enrollment was 29days, and patients failed an average of 3.2 lines of treatment for GVHD.Organ involvement was 60% skin, 87% gastrointestinal, and 38% liver. Atday 28, overall response (OR), defined as organ improvement of at leastone stage without worsening in any other, was 64%; 17% of patients hadstable disease or mixed response; and 19% experienced progression. Bygrade, 28 day OR was 67% for B, 75% for C, and 58% for D. Response byorgan was recorded, with 47% of skin, 23% of GI, and 39% of liver GVHDcompletely responding (stage 0) within the first 28 days of treatment.

Overall survival through study duration (day 100) was 62%. Achieving anOR at day 28 resulted in a significantly higher probability of survivalwhen compared to patients who progressed within the first 28 days (76%vs 9%, p<0.05). The MSCs were well tolerated and there was no evidenceof ectopic tissue formation.

These results demonstrate that preparations of the present invention aresurprisingly therapeutically effective, even when expanded on a large(clinical) scale.

Example 43 Therapeutic Efficacy of Clinical Scale Preparations

Pediatric patients (<18 years) with grade B-D SR-GVHD were randomized toreceive MSCs from a clinical scale preparation produced by the methoddetailed in Example 1 through Example 26 or placebo, in addition tostandard of care, including institutionally selected second line agent.Patients received 8 therapeutic doses of 2×10⁶ cells/kg for 4 weeks (orvolume equivalent for placebo), with 4 more infusions weekly in patientswho had a response. The primary endpoint was durable complete response(CR>28 days); secondary endpoints included incidence of CR, overallresponse (OR=CR+PR), progression through 100 days, and survival.

Twenty-eight children were randomized to MSC treatment (50% male, 79%Caucasian) or placebo (71% male, 71% Caucasian), with a median age of 7years (range 1-15) and 10 years (range 1-18), respectively. The dominanttransplant graft was cord blood (71% MSC, 57% placebo), with mostlyunrelated donors (93% vs 79%, respectively). The median duration ofaGVHD prior to enrollment was 20 days for MSC treatment and 8 days forplacebo (p<0.05). At baseline, aGVHD grades B:C:D were 3:8:3 for botharms. For MSC treatment, organ involvement was 64% skin, 43% GI, and 36%liver. For placebo patients, organ involvement was 57% skin, 79% GI, and29% liver. Durable CR was 64% for MSC treatment and 43% for placebo. CRfor MSC treatment vs placebo, respectively, was 36% vs 21% at day 28,57% vs 21% at day 42, and 64% vs 29% at day 100. OR for MSC treatment vsplacebo, respectively, was 64% vs 34% at day 28, 64% vs 50% at day 42,and 71% vs 50% at day 100. Progression for MSC treatment vs placebo,respectively, was 14% vs 50% at day 28, 29% vs 43% at day 42, and 21% vs43% at day 100.

Survival for MSC treatment vs placebo, respectively, was 93% vs 86% atday 28, 79% vs 79% at day 42, and 79% vs 50% at day 100. The median timeto CR was 25 days vs 63 days. The 25% percentile of the survivalfunction after study start was 139 days for MSC treatment and 50 daysfor placebo.

These results demonstrate that preparations of the present invention aresurprisingly therapeutically effective, even when expanded on a large(clinical) scale.

Example 44 Therapeutic Efficacy of Clinical Scale Preparations

244 patients with grade B-D SR-GVHD (skin involvement n=144, GIinvolvement n=179, liver involvement n=61) were randomized to receiveMSCs from a clinical scale preparation produced by the method detailedin Example 1 through Example 26 (n=163) or placebo (n=81), in additionto standard of care, including institutionally selected second lineagent. Patients received 8 therapeutic doses of 2×10⁶ cells/kg for 4weeks (or volume equivalent for placebo), with 4 more infusions weeklyin patients who responded. The primary endpoint was durable completeresponse (CR>28 days).

For the MSC treatment and placebo arms, respectively, the grades of GVHDat entry were B (22% vs. 26%), C (51% vs. 58%), and D (27% vs 16%). Therespective durable CR rates were 35% vs. 30% (p=0.3) in the ITTpopulation and 40% vs. 28% (p=0.08) in the per protocol population.Patients with GVHD affecting all 3 organs had an overall complete orpartial response rate of 63% vs. 0% (n=22, p<0.05) at day 28. Patientstreated with MSCs had less progression of liver GVHD at weeks 2 and 4respectively (32% vs 59%, p=0.05; and 37% vs. 65%, p=0.05).

The overall response (OR) results at day 100 were 82% in the MSC-treatedgroup and 73% in the placebo group.

The partial response results at day 100 for the MSC-treated group andplacebo group, respectively, were: Skin, 78% and 77%; Liver, 76% and47%; Gut, 82% and 68%.

These results demonstrate that preparations of the present invention aresurprisingly therapeutically effective, even when expanded on a large(clinical) scale.

Example 45 Therapeutic Efficacy of Clinical Scale Preparations

Patients with grades II-IV GVHD were randomized to receive human MSCsderived from a clinical scale preparation produced by the methoddetailed in Example 1 through Example 26 in 2 therapeutic doses ofeither 2 or 8 million MSCs/kg in combination with corticosteroids. Thestudy evaluated induction of response to MSC therapy, and overallresponse of aGVHD by day 28. Thirty-one patients were evaluated: 21males, 10 females; median age 52 years (range: 34-67). Twenty-onepatients had grade II, 8 had grade HI, and 3 had grade IV aGVHD.Ninety-four percent of patients had an initial response to MSC therapy(77% complete response and 16% partial response).

These results demonstrate that preparations of the present invention aresurprisingly therapeutically effective, even when expanded on a large(clinical) scale.

Example 46 Variant Preparations

Using the methodologies generally set forth in Example 1-Example 41, amanufacturing facility produced multiple preparations of MSCs. Of the 27independent preparations manufactured that were fully analyzed (until afailure was identified), labeled according to the manufacturingprotocol, and successfully passed sterility and visual appearance tests,14 preparations met the following criteria:

CD 166+: ≥95% CD 105+ ≥95% CD 45− ≥99.25%   Post-thaw viability: ≥75%Residual BSA ≥10 μg/ml Residual Trypsin ≤30 μg/ml TNFRI 108-368 pg/mlIL2-Ra ≥30% inhibition

Of the 13 that failed to meet one or more criteria, 8 failed todemonstrate potency based upon the IL-2Rα test, 4 failed to demonstratepotency based upon TNF R1 expression, and 2 had excess residual trypsinlevels. One of the preparations failed the TNFRI assay also failed theIL-2Rα assay.

These results demonstrate that MSC preparations can show heterogeneity,even when standardized procedures are followed. Thus, the skilledartisan should now recognize that MSC preparations in the art thatappear equivalent based one or even several criteria may, in fact, haveimportant phenotypic or genotypic differences between them and may notdemonstrate the potency that characterizes the MSC preparations of thepresent invention. Moreover, the MSC preparations of the presentinvention can be distinguished from many of the preparations in theprior art upon complete characterization as taught here.

Example 47 Variant Preparations

At another manufacturing facility (i.e. different facility than inExample 46), the methodologies set forth in Example 1-Example 41 weregenerally followed. Greater than 95% of the lots met the criteria ofExample 46. However, one preparation failed post-thaw viability test andthe TNFR1 assay for potency. Another preparation failed the residualtrypsin analysis, having a value of 38.1 μg/mL. These resultsdemonstrate the importance of adding a screening step to themanufacturing methods, even when highly reproducible techniques havebeen achieved.

The citations provided herein are hereby incorporated by reference forthe cited subject matter.

The presently described technology is now described in such full, clear,concise and exact terms as to enable any person skilled in the art towhich it pertains, to practice the same. It is to be understood that theforegoing describes preferred embodiments of the technology and thatmodifications may be made therein without departing from the spirit orscope of the invention as set forth in the appended claims.

What is claimed is:
 1. A method of treating graft-versus-host-disease (GVHD) in a human in need thereof comprising administering a therapeutic amount of culture-expanded mesenchymal stem cells (MSCs) comprising (i) less than 0.75% CD45+ cells, (ii) at least 95% CD105+ cells, and (iii) at least 95% CD166+ cells, wherein the MSC are capable of inhibiting IL2Rα expression by CD3/CD28-activated peripheral blood mononuclear cells PBMCs by at least 30% relative to a control.
 2. The method of claim 1, wherein the GVHD is steroid-refractory.
 3. The method of claim 1, wherein the MSC were cultured-expanded from cryopreserved MSC.
 4. The method of claim 1, wherein the MSCs express at least 13 pg TNFR1 per million MSCs.
 5. The method of claim 1, wherein the MSC express about 13 pg to about 44 pg TNFRI per million MSCs.
 6. The method of claim 1, wherein said culture-expansion comprises at least 20 population doublings.
 7. The method of claim 1, wherein said culture-expansion comprises at least 30 population doublings.
 8. The method of claim 1, wherein the MSC are autologous.
 9. The method of claim 1, wherein the MSC are allogeneic.
 10. The method of claim 1, wherein the MSC are administered at a dose of 2×10⁶ cells/kg.
 11. The method of claim 10, wherein the MSC are administered biweekly.
 12. The method of claim 11, further comprising subsequent weekly infusions.
 13. The method of claim 1, wherein the MSC are obtained from cord blood.
 14. The method of claim 1, wherein the GVHD is grade II GVHD.
 15. The method of claim 1, wherein the GVHD is grade III or grade IV GVHD.
 16. A method of treating an autoimmune disease in a human in need thereof comprising administering a therapeutic amount of culture-expanded mesenchymal stem cells (MSCs) comprising (i) less than 0.75% CD45+ cells, (ii) at least 95% CD105+ cells, and (iii) at least 95% CD166+ cells, wherein the MSC are capable of inhibiting IL2Rα expression by CD3/CD28-activated PBMCs by at least 30% relative to a control.
 17. The method of claim 16, wherein the autoimmune disease is inflammatory bowel disease (IBD).
 18. The method of claim 17, wherein the IBD is Crohn's disease.
 19. The method of claim 16, wherein the autoimmune disease is multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, uveitis, autoimmune thyroid disease, scleroderma, Graves' Disease, lupus, autoimmune lymphoproliferative disease (ALPS), demyelinating disease, autoimmune encephalomyelitis, autoimmune gastritis (AIG), or autoimmune glomerular disease.
 20. A method of treating asthma in a human in need thereof comprising administering a therapeutic amount of culture-expanded mesenchymal stem cells (MSCs) comprising (i) less than 0.75% CD45+ cells, (ii) at least 95% CD105+ cells, and (iii) at least 95% CD166+ cells, wherein the MSC are capable of inhibiting IL2Rα expression by CD3/CD28-activated PBMCs by at least 30% relative to a control. 